Methods for producing sialyloligosaccharides in a dairy source

ABSTRACT

The present invention provides methods for producing sialyloligosaccharides in situ in dairy sources and cheese processing waste streams, prior to, during, or after processing of the dairy source during the cheese manufacturing process. The methods of the present invention use the catalytic activity of α(2-3) trans-sialidases to exploit the high concentrations of lactose and α(2-3) sialosides which naturally occur in dairy sources and cheese processing waste streams to drive the enzymatic synthesis of α(2-3) sialyllactose. α(2-3) sialyloligosaccharides produced according to these methods are additionally encompassed by the present invention. The invention also provides for recovery of the sialyloligosaccharides produced by these methods. The invention further provides a method for producing α(2-3) sialyllactose. The invention additionally provides a method of enriching for α(2-3) sialyllactose in milk using transgenic mammals that express an α(2-3) trans-sialidase transgene. The invention also provides for recovery of the sialyllactose contained in the milk produced by this transgenic mammal either before or after processing of the milk. Transgenic mammals containing an α(2-3) trans-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue are also provided by the invention.

1. INTRODUCTION

This invention relates to methods for producing α(2-3)sialyloligosaccharides in a dairy source or cheese processing wastestream by contacting the dairy source or cheese processing waste streamwith a catalytic amount of at least one α(2-3) trans-sialidase. Inpreferred embodiments, the methods of the invention are applied toproduce α(2-3) sialyllactose in a dairy source or cheese processingwaste stream. Methods for isolating the α(2-3) sialyloligosaccharidesproduced according to the methods of the invention are also provided.The invention additionally relates to a method for producing α(2-3)sialyllactose in milk using a transgenic mammal containing an α(2-3)trans-sialidase encoding sequence operably linked to a regulatorysequence of a gene expressed in mammary tissue.

2. BACKGROUND OF THE INVENTION

2.1. Sialyloligosaccharides in Cheese Waste Streams

Whey is a major by-product of cheese manufacturing, which, forenvironmental reasons, presents a difficult waste disposal problem. Inthe United States alone, fluid whey is being produced at a rate of about62.6 billion pounds annually. Whey is typically composed of about 5 wt.% lactose, 1 wt. % protein and about 0.5 wt. % salts, where the balanceof the mixture is water. A major effort by many cheese making countriesis presently underway to develop uses for this commodity, which formerlywas considered a cheese processing waste product.

Although the protein concentrate obtained by ultrafiltration of whey hasbecome a valuable commodity in the food industry and has foundapplications in animal feed, fertilizer, fermentation, and food filler,the majority of the resulting lactose-rich ultrafiltered permeate isstill considered a disposable fraction.

Recently, several sialyloligosaccharides have been found to havevaluable application as pharmaceutics. See, e.g. U.S. Pat. No. 5,270,462to Shimatani et al. Sialyllactose has been shown to neutralizeenterotoxins of various pathogenic microbes including Escherichia coli,Vibrio cholerae and Salmonella. See, e.g. U.S. Pat. No. 5,330,975 toHiroko et al. It has also been shown that α(2-3) sialyllactose(α-Neu5Ac-(2-3)-Gal-β-(1-4)-Glc) interferes with colonization ofHelicobacter pylori and thereby prevents or inhibits gastric andduodenal ulcers. See e.g. U.S. Pat. No. 5,514,660 to Zopf et al.Sialyllactose has additionally been proposed to inhibit immune complexformation by disrupting occupancy of the Fc carbohydrate binding site onIgG and to be useful in treating arthritis. See, e.g. U.S. Pat. No.5,164,374 to Rademacher et al.

To date, commercially available sialyloligosaccharides have been veryexpensive due to their low quantity in natural sources. For example,α(2-3) sialyllactose and α(2-6) sialyllactose isolated from bovinecolostrum, is sold for $75.60 and $83.30 per milligram, respectively(Sigma Chemical Company, 1997).

A focused effort has been directed toward harvestingsialyloligosaccharides from the vast supply of whey made available as acheese processing waste product. Processes for isolatingsialyloligosaccharides have utilized such techniques as ultrafiltration,ion-exchange resins and phase partition chemistry. U.S. Pat. No.4,001,198 to Thomas and U.S. Pat. No. 4,202,909 to Pederson; U.S. Pat.No. 4,547,386 to Chambers et al.; U.S. Pat. No. 4,617,861 to Armstrong;U.S. Pat. Nos. 4,971,701 and 4,855,056 to Harju et al.; U.S. Pat. No.4,968,521 to McInychyn; U.S. Pat. No. 4,543,261 to Harmon et al.; U.S.Pat. Nos. 5,118,516 and 5,270,462 to Shimatani; J P Kokai 01-168,693; JP Kokai 03-143,351; J P Kokai 59-184,197; J P Kokoku 40-1234; J P Kokai63-284,199 and Japanese Patent Publication No. 21234/1965, each of whichis herein incorporated by reference in its entirety. Yields of up to 6grams of α(2-3) sialyllactose sialyloligosaccharide per kilogram ofcheese processing waste stream have been reported. U.S. Pat. No.5,575,916 to Brian et al. which is herein incorporated by reference inits entirety.

2.2. Sialidases and Sialyltransferases

Sialic acids are 9-carbon carboxylated sugars which generally occur asthe terminal monosaccharides in oligosaccharide chains. In mammaliancells, sialic acids are most frequently linked to β-galactose with anα(2-3) linkage, and to N-acetylglucosamine and N-acetylgalactosaminewith an α(2-6) linkage. Cross et al., 1993, Annu. Rev. Microbiol.47:385-411.

Sialidases catalyze the removal of sialic acid residues from theoligosaccharide chain. Due to the wide variety of substitutions whichmay occur at various carbons of the sialic acid molecules, there are atleast 39 different species of sialic acids. Colli, W., 1993, FASEB J.7:1257-1264. Generally, sialidases exhibit substrate specificity forspecific forms of sialic acid linkages. Viral sialidases cleave α(2-3)glycosidic bonds more efficiently than α(2-6) bonds, but bacterialsialidases are not as specific. Cross et al., 1993, Annu. Rev.Microbiol. 47:385-411 (citing Corfield et al. 1982, Sialic Acids:Chemistry, Metabolism and Function, Vol. 10, New York: Springer-Verlag,pp. 195-261). At low enzyme concentrations, bacterial sialidases exhibita preference for cleaving α(2-3) or α(2-6) glycosidic bonds. Cross etal., 1993, Annu. Rev. Microbiol. 47:385-411.

CMP-sialyltransferases catalyze the transfer of cytidinemonophosphate-sialic acid (CMP-sialic acid) residues to acceptormolecules. Although many sialidases exhibit at least some substratespecificity, CMP-sialyltransferases act on specific substrates.Mammalian CMP-sialyltransferases are generally found in the Golgi,however, there is evidence that there may be cell-surface associatedCMP-sialyltransferases as well. Cross et al., 1993, Annu. Rev.Microbiol. 47:385-411 (citing Roth et al., 1971, J. Cell Biol.51:536-547; Shur, 1991, Glycobiology 1:563-575; Yogeeswaran et al.,1974, Biochem. Biophys. Res. Commun. 59:591-599).

2.3. Trypanosoma Cruzi α(2-3)-Trans-Sialidase

Trypanosoma cruzi (Order Kinetoplastida) is the intracellular parasiteresponsible for Chagas diseage, throughout Iberoamerican countries.Chagas disease primarily affects nerve and muscle cells. One seriousmanifestation of Chagas disease is a chronic progressive fibroticmyocarditis. Colli, 1993, FASEB J. 7:1257-1264. Approximately 16-18million people are infected with T. cruzi. Colli, 1993, FASEB J.7:1257-1264.

T. cruzi invades a broad range of host cells, and a considerable amountof research has focused on the surface molecules in order to determinewhich molecules may be involved in parasite/host interaction. Colli,1993, FASEB J. 7:1257-1264. One surface molecule which has generated agreat deal of interest is the α(2-3)-trans-sialidase. This molecule hasthe capability of catalyzing both the removal of sialic acid from adonor saccharide-containing molecule (sialidase activity) and catalyzingthe transfer of the sialic acid to an acceptor saccharide-containingmolecule (trans-sialidase activity). Schankman et al., 1992, J. Exp.Med. 175:567-575. The gene encoding T. cruzi trans-sialidase has beencloned and characterized at the molecular level.

The T. cruzi α(2-3) trans-sialidase catalyzes the transfer of sialicacid from a donor terminal β-galactosyl sialoglycoconjugate to aterminal β-galactose on an acceptor molequle, Collit W., 1993, FASEB J.7:1257-1264. T. cruzi α(2-3) trans-sialidase does not use CMP-sialicacid as a substrate and prefers sialyl α(2-3)-linked to β-galactosylresidues as sialic acid donor molecules over sialyl α(2-6)-, α(2-8)-,and α(2-9)-linked sialic acids. Schenkman et al., 1994, Annu. Rev.Microbiol. 48:499-523. Furthermore, T. cruzi α(2-3) trans-sialidasecannot use free sialic acid as a substrate. Vandekerckhove et al. 1992,Glycobiology 2:541-548. The T. cruzi α(2-3) trans-sialidase has a broadpH optimum centered at 7.0. Cross et al., 1993, Annu Rev. Microbiol.47:385-411.

More detailed analysis of the α(2-3) trans-sialidase has revealed thatthe amino-terminal portion of the protein is responsible for the α(2-3)trans-sialidase activity. Campetella et al., 1994, Mol. Biochem.Parasitol. 64:337-340; Schenkman et al., 1994, J. Biol. Chem.269:7970-7975. It has also been determined that there are at least twocritical amino acid residues: Tyr³⁴² and Pro²³¹ of the α(2-3)trans-sialidase appear to be required for full α(2-3) trans-sialidaseactivity. Cremona et al., 1995, Gene 160:123-25. The importance ofTyr³⁴² is demonstrated by the fact that naturally occurring variants ofthe T. cruzi α(2-3) trans-sialidase which have a Tyr³⁴²→Hissubstitution, lack α(2-3) trans-sialidase activity. Uemura et al., 1992,EMBO J. 11:3837-3844.

Trans-sialidase activity has also been discovered in Trypanosoma brucei,the causative agent of African Sleeping Sickness, Endotrypanum spp. andin Pneumocystis carinii. Like the T. cruzi α(2-3) trans-sialidase, theT. brucei trans-sialidase has a pH optimum of 7.0. However, unlike theT. cruzi trans-sialidase, which is expressed during the trypomastigotestage, the T. brucei trans-sialidase appears to be expressed only duringthe procyclic stage of the parasite life cycle, when the parasiteresides in the midgut of its insect vector (Glossina spp., the “tsetsefly”). Cross et al., 1993, Annu Rev. Microbiol. 47:385-411.

2.4. Sialyllactose Production

A variety of methods for enzymatically producing sialylatedoligosaccharides have been described.

U.S. Pat. No. 5,374,541 to Wong et al., describes a method for producingsialyloligosaccharides. According to this method, β-galactosidase isused to form β-galactosyl glycosides in the presence of CMP-sialic acidand α(2-3)- or α(2-6)-CMP-sialyltransferases to form sialylatedoligosaccharides. This method does not use α(2-3) trans-sialidase.

U.S. Pat. No. 5,409,817 to Ito et al., discloses a three enzyme processfor producing α(2-3) sialylgalactosides. According to this process,CMP-sialyltransferases transfer sialic acid from CMP-sialic acid toacceptor molecules, these acceptor molecules become donor molecules forTrypanosoma cruzi α(2-3) trans-sialidase, and CMP-sialic acid isregenerated in the system through the action of CMP-sialic acidsynthetase and added free sialic acid.

The process described in U.S. Pat. No. 5,409,817 to Ito et al.specifically requires the addition of free sialic acid. The free sialicacid is converted to CMP-sialic acid by CMP-sialic acid synthetase, andthe sialic acid moiety is transferred to an acceptor molecule byCMP-sialyltransferase. According to the disclosure of Ito et al., theformation of these sialylated acceptor molecules is required to drivethe α(2-3) trans-sialidase reaction forward.

In addition to free sialic acid, the method of Ito et al., also requiresthe presence of three enzymes including CMP-sialic acid synthetase andCMP-sialyltransferase. Further, dairy sources and cheese processingwaste streams do not contain CMP-sialic acid synthetase.

2.5. Expression of Transgenes in Milk

Numerous foreign proteins have successfully been transgenicallyexpressed in the milk of livestock. Most of this work has focused on theexpression of proteins which are foreign to the mammary gland. Colman,A., 1996, Am. J. Clin. Nutr. 63:639S-645S. To date, milk specificexpression of transgenic livestock has been achieved through operablylinking regulatory sequences of milk-specific protein genes to thetarget protein-encoding gene sequence, microinjecting these geneticconstructs into the pronuclei of fertilized embryos, and implanting theembryos into recipient females. See e.g. Wright et al., 1991,Biotechnology (NY) 9:830-834; Carver et al., 1993, Biotechnology (NY)11:1263-1270; Paterson et al., 1994, Appl. Microbiol. Biotechnol.40:691-698. Proteins that have been successfully expressed in the milkof transgenic animals, include: α1-antitrypsin (Wright et al., 1991,Biotechnology (NY) 9:830-834; Carver et al., 1993, Biotechnology (NY)11:1263-1270); Factor IX (Clark et al., 1989, Biotechnology (NY)7:487-492); protein C (Velander et al., 1992, Proc. Natl. Acad. Sci.USA, 89:12003-12007); tissue plasminogen activator (Ebert et al., 1991,Biotechology (NY) 9:835-838); and fibrinogen. While most of thesetransgenes express proteins that supplement the composition of milk,very few, if any of the expressed proteins interact directly with thecomponents of milk to alter the natural milk composition. There is aneed for methods providing for the large scale production of α(2-3)sialyloligosaccharides, such as α(2-3) sialyllactose, which havecommercial and/or therapeutic valve.

3. SUMMARY OF THE INVENTION

The present invention greatly advances the field of commercialproduction of sialyloligosaccharides by providing methods for producingsialyloligosaccharides in situ in dairy sources and cheese processingwaste streams. The methods of the invention have particular applicationsin producing α(2-3) sialyllactose in a dairy source prior to, during, orafter processing of the dairy source during the cheese manufacturingprocess, thereby greatly increasing the recoverable yield of α(2-3)sialyllactose from the dairy source.

Dairy sources and cheese processing waste streams are known to containhigh concentrations of lactose and numerous α(2-3) sialosides, such as,for example, κ casein, and the gangliosides. Applicants are the first toprovide a method for producing α(2-3) sialyllactose in a dairy source ora cheese processing waste stream. More specifically, the method of thepresent invention uses the catalytic activity of α(2-3) trans-sialidasesto exploit the high concentrations of lactose and α(2-3) sialosideswhich naturally occur in dairy sources, to drive the enzymatic synthesisof α(2-3) sialyllactose. This catalytic activity does not require thepresence of CMP-sialic acid synthetase, CMP-sialyltransferase and/orfree siallic acid to drive the sialylation of α(2-3) sialyllactose andother α(2-3) sialyloligosaccharides.

Accordingly, the invention provides a novel method for producing α(2-3)sialyloligosaccharides, and specifically, α(2-3) sialyllactose(α-Neu5Ac-(2-3)-Gal-β-(1-4)-Glc), in a dairy source or cheese processingwaste stream by catalyzing the sialidation of lactose (Gal-β-(1-4)-Glc).In specific embodiments, the method of the invention is applied to thedairy source prior to or during processing. In another specificembodiment, the method of the present invention is applied afterprocessing of the dairy source (e.g. to a cheese processing wastestream).

The present invention provides a method for producingsialyloligosaccharides in a dairy source. This method comprisescontacting a catalytic amount of least one α(2-3) trans-sialidase with adairy source to form a dairy/trans-sialidase mixture and incubating thedairy/trans-sialidase mixture under conditions suitable for α(2-3)trans-sialidase activity. α(2-3) sialyloligosaccharides producedaccording to this method are additionally encompassed by the presentinvention. The invention also provides for recovery of thesialyloligosaccharides contained in the incubated dairy/trans-sialidasemixture or alternatively, in compositions formed after processing of theincubated dairy/trans-sialidase mixture (e.g. a cheese processing wastestream), using techniques which include, but are not limited to,ultrafiltration, diafiltration, nanofiltration, electrodialysis, phasepartitioning and ion exchange chromatography.

The present invention also provides a method for producingsialyloligosaccharides in a cheese processing waste stream. This methodcomprises contacting a catalytic amount of at least one α(2-3)trans-sialidase with a cheese processing waste stream to form a wastestream/trans-sialidase mixture and incubating the wastestream/trans-sialidase mixture under conditions suitable for α(2-3)trans-sialidase activity. α(2-3) sialyloligosaccharides producedaccording to this method are additionally encompassed by the presentinvention. The invention also provides for recovery of thesialyloligosaccharides contained in the incubated dairy/trans-sialidasemixture using techniques which include, but are not limited to,ultrafiltration, diafiltration, nanofiltration, electrodialysis, phasepartitioning and ion exchange chromatography.

The invention further provides a method for producing α(2-3)sialyllactose. This method comprises contacting a catalytic amount of atleast one α(2-3) trans-sialidase with lactose and an α(2-3)sialyloligosaccharide, in the absence of CMP-sialyltransferase, to forma mixture, and incubating this mixture under conditions suitable forα(2-3) trans-sialidase activity. α(2-3) sialyllactose produedd accordingto this method are additionally encompassed by the present invention.The invention also provides for recovery of the sialyllactose containedin this incubated mixture using techniques which include, but are notlimited to, ultrafiltration, diafiltration, nanofiltration,electrodialysis, phase partitioning and ion exchange chromatography.

The invention additionally provides a method of enriching for α(2-3)sialyllactose in milk using transgenic mammals that express an α(2-3)trans-sialidase transgene. According to this method, a transgenecomprising an α(2-3) trans-sialidase encoding sequence is operablylinked to a regulatory sequence of a gene expressed in mammary tissueand this α(2-3) trans-sialidase/regulatory sequence transgene is thenintroduced into the germline of a mammal to produce a transgenic mammal.The milk produced by a transgenic mammal demonstrating α(2-3)trans-sialidase activity in mammary tissue, contains enriched α(2-3)sialyllactose concentrations. The invention also provides for recoveryof the sialyllactose contained in the milk produced by this transgenicmammal either before or after processing of the milk. Transgenic mammalscontaining an α(2-3) trans-sialidase encoding sequence operably linkedto a regulatory sequence of a gene expressed in mammary tissue are alsoprovided by the invention. Significantly, a dairy source, cheeseprocessing waste stream, and transgenic mammal can be used to produceenriched concentrations of α(2-3) sialyllactose.

As used herein, “trans-sialidase” refers to a compound that catalyzesthe transfer of a sialic acid from one saccharide-containing molecule(e.g. oligosaccharide, polysaccharide, glycoprotein or glycolipid) toanother saccharide-containing molecule and which does not require thepresence of free sialic acid, CMP-sialic acid, synthetase and/orCMP-sialyltransferase in the reaction mixture for its activity.

As used herein, “trans-sialidase activity” refers to the catalyticreaction in which an enzyme catalyzes the removal of a sialic acid fromone saccharide-containing molecule and the transfer of the sialic acidto another saccharide-containing molecule, covalently attaching thesialic acid to the acceptor molecule through a glycosidic bond.

As used herein, a “catalytic amount” of α(2-3) trans-sialidase enzymerefers to the quantity of enzyme sufficient to cause the transfer of asialic acid from one saccharide-containing molecule to anothersaccharide-containing molecule.

As used herein, “conditions suitable for trans-sialidase activity”encompass appropriate conditions (e.g. temperature, pH and incubationtime) sufficient to permit the enzymatic removal of a sialic acid fromone saccharide-containing molecule and the transfer of the sialic acidto another saccharide-containing molecule.

As used herein, “α(2-3) sialyloligosaccharides” refer to sugars in whicha sialic acid is covalently attached to the 3′ carbon of a β-galactosemoiety through a glycosidic bond. In the methods of the presentinvention, α(2-3) sialyloligosaccharides encompass saccharides with anyform of sialic acid covalently attached to the 3′-β-galactose.

As used herein, “dairy source” refers to a product of lactation in amammal, a substance made by the product, or a byproduct thereof. As usedherein, “dairy source” includes, but is not limited to, milk, colostrum,a cheese processing mixture, and a composition simulating milk.

As used herein, a “cheese processing mixture” is a compilation ofingredients of dairy processing at any stage during dairy processing(e.g. pasteurization, fermentation, or cheese manufacture) other thanthe cheese processing waste stream.

As used herein, “a composition simulating milk” is a solution lackingone or more of CMP-sialyltransferase, CMP-synthetase and/or free sialicacid, but which contains at least α(2-3) sialosides to act as donors forthe trans-sialidase, lactose and, optionally, appropriate bufferingagents to maximize the activity of the α(2-3) trans-sialidase when it isadded to the solution.

As used herein, “cheese processing waste stream” refers to a byproductof cheese manufacture and includes, but is not limited to, whole whey,demineralized whey permeate, the regeneration stream from demineralizedwhey permeate, whey permeate, crystallized lactose, spray dried lactose,whey powder, edible lactose and lactose. Whey containing sialic acids,is a byproduct obtained when cheese or rennet casein is produced frommilks such as cow milk, goat milk, and sheep milk. For example acidwhey, is generated by separating the solids when gkim milk is coagulatedto form cottage cheese. Acid whey is characterized by a high lactic acidcontent. When cheese is prepared from whole milk, the remaining liquidis sweet whey which can be further processed by evaporation to form drywhey powder. Sweet whey can also be dried, demineralized and evaporatedto form demineralized whey permeate. Sweet whey can also be subjected toultrafiltration to generate both a whey permeate and a whey proteinconcentrate. Whey permeate can be further processed by crystallizinglactose to form both lactose and a mother liquor. The mother liquorresulting from crystallizing lactose from a whey permeate is known inthe art as “Delac.”

The α(2-3) trans-sialidase used according to the method of the presentinvention encompasses Kinetoplastid trans-sialidases, trans-sialidasesderived from Trypanosoma, Endotrypanum, and Pneumocystis, and includestrans-sialidases of Trypanosoma cruzi, Trypanosoma brucei, Endotrypanumspp. and Pneumocystis carinii. Trans-sialidases that may be usedaccording to the method of the present invention are further definedinfra in Section 5.1.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Complete nucleotide sequence (SEQ ID NO:1) of Trypanosoma cruzi(Genbank D50685) α(2-3) trans-sialidase.

FIG. 2. Deduced amino acid sequence (SEQ ID NO: 2) of Trypanosoma cruzi(Genbank D50685) α(2-3) trans-sialidase.

FIG. 3. Nucleotide sequence (SEQ ID NO: 3) of a functional Trypanosomacruzi α(2-3) trans-sialidase lacking the amino acid repeats (GenbankL26499).

FIG. 4. Deduced amino acid sequence (SEQ ID NO: 4) of a functionalTrypanosoma cruzi α(2-3) trans-sialidase lacking the amino acid repeats(Genbank L26499).

FIG. 5. Effect of pH on α(2-3) sialyllactose enrichment in mozzarellawhey. The α(2-3) sialyllactose concentration (μg/mL) is shown as afunction of time of incubation of 0.1% α(2-3) trans-sialidase lysate at25° C. Squares represent pH 4.0; open diamonds represent pH 5.0; circlesrepresent pH 6.0; triangles represent pH 7.0; crossed squares representpH 8.0; and shaded diamonds represent pH 9.0. α(2-3) sialyllactoseenrichment was observed at all pHs tested, with only minimal enrichmentobserved after 20 minutes at pH 4.0.

FIG. 6. Enrichment of α(2-3) sialyllactose in skim milk. Theconcentration of α(2-3) sialyllactose (μg/mL) is shown as a function oftime of incubation with 0.1% trans-sialidase lysate at 22° C.

FIGS. 7A-B. Enrichment of α(2-3) sialyllactose in mozzarella whey. Theconcentration of α(2-3) sialyllactose (μg/mL) is shown as a function oftime of incubation of 0.1% α(2-3) trans-sialidase lysate at 25° C. (FIG.7A) and 23° C. (FIG. 7B).

FIG. 8. Enrichment of α(2-3) sialyllactose in Swiss cheese whey. Theconcentration of α(2-3) sialyllactose (μg/ml) is shown as a function oftime of incubation of 0.1% α(2-3) trans-sialidase lysate at 23° C. over43 hours.

FIG. 9. Enrichment of α(2-3) sialyllactose in a solution containing 20mg/ml lactose, 5 mg/ml κ-casein at 23° C., over 22 hours.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods for producing sialyloligosaccharides ina dairy source, particularly, α(2-3) sialyllactose, by contacting acatalytic amount of α(2-3) -trans-sialidase with a dairy source to forma dairy/trans-sialidase mixture and incubating this mixture underconditions suitable for α(2-3) trans-sialidase activity. The inventionalso relates to methods for recovering α(2-3) sialylatedoligosaccharides from this incubated dairy/trans-sialidase mixture, oralternatively, from compositions formed after processing of thedairy/trans-sialidase mixture (e.g. a cheese processing waste stream).In a specific embodiment, α(2-3) sialyllactose is recovered from theprocessed composition by ultrafiltration and ion exchangechromatography.

The invention additionally provides for methods of producing α(2-3)sialyloligosaccharides in a cheese processing waste stream by contactinga catalytic amount of trans-sialidase with a cheese processing wastestream to form a waste stream/trans-sialidase mixture and incubatingthis mixture under conditions suitable for α(2-3) trans-sialidaseactivity. The invention also relates to methods for recovering α(2-3)sialyloligosaccharides from this incubated waste stream/trans-sialidasemixture.

The methods of the present invention can be used to produce α(2-3)sialyloligosaccharides in any reaction mixture containing α(2-3)sialylated saccharide compositions (e.g. oligosaccharides,polysaccharides, glycoproteins, and glycolipids) and lactose. Startingmaterials may therefore be derived from all dairy sources (e.g. humanand animal milk, whey and colostrum) or alternatively, a mixture oflactose and α(2-3) sialylated saccharide compositions which simulates adairy source.

5.1. α(2-3) Trans-Sialidase

The α(2-3) trans-sialidase used according to the method of the inventionis an α(2-3) trans-sialidase, or derivative (including fragments orfusion proteins), or analog thereof, which is able to catalyze theremoval of sialic acid from one saccharide-containing molecule andcatalyze the transfer of the sialic acid to a secondsaccharide-containing moleculoe

The α(2-3) trans-sialidases that may be used according to the method ofthe invention include, but are not limited to, a Kinetoplastid α(2-3)trans-sialidase from a species of the genera Trypanosoma, Endotrypanum,and Pneumocystis, such as, for example, Trypanosoma cruzi α(2-3)trans-sialidase, T. brucei α(2-3) trans-sialidase (Pontes de Carvalho etal., 1993, J. Exp. Med. 177:465-474), Pneumocystis cariniitrans-sialidase (L. Trimbal, N. Pavia & M. E. A. Pereira, unpublishedinformation as cited in Schenkman et al., 1994, Annu. Rev. Microbiol.48:499-523), and Endotrypanum spp. trans-sialidase (Medina-Acosta etal., 1994, Mol. Biochem Parasitol. Nucleic acid sequences oftrans-sialidases are known (for example, Genbank Sequence L26499,SPTREMBL:Q26964 (Uemura), SPTREMBL:Q26965 (Uemura), SPTREMBL:Q26966(Uemura), SPTREMBL:Q26969 (Cremona et al.), Genbank D50685 (Uemura).

In specific embodiments, a polypeptide consisting of or comprising afragment of at least 50 (continuous) amino acids of an α(2-3)trans-sialidase are used according to the method of the invention. Inother embodiments, the fragment consists of at least 100, 150, 200, 250,300, 350, 400, 450, 500, or 550 amino acids of the α(2-3)trans-sialidase. In further specific embodiments, such fragments are notlarger than 500, 400, 300, 200 or 100 amino acids. Derivatives oranalogs of a α(2-3) trans-sialidase, include but are not limited to,those molecules that catalyze the transfer of sialic acid from onesaccharide-containing molecule (e.g. a oligosaccharide, polysaccharide,glycoprotein, or glycolipid)) to another saccharide-containing moleculeand that are encoded by a DNA sequence that hybridizes to the complementof a DNA sequence that encodes a α(2-3) trans-sialidaser such as, forexample, those listed above, under high stringency, moderately highstringency, or low stringency conditions.

By way of example and not limitation, procedures using conditions of lowstringency are as follows (see also Shilo and Weinberg, 1981, Proc.Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA arepretreated for 6 h at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations arecarried out in the same solution with the following modifications: 0.02%PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol)dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 40° C., and thenwashed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and reexposed to film.Other conditions of low stringency which may be used are well known inthe art.

By way of example and not limitation, procedures using conditions ofhigh stringency are as follows: prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C. in prehybridization mixture containing 100μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe.Washing of filters is done at 37° C. for 1 h in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C. for 45 min before autoradiography. Otherconditions of high stringency which may be used are well known in theart.

By way of example and not limitation, procedures using conditions ofmoderately high stringency are as follows: filters containing DNA arepretreated for 6 hours to overnight at 55° C. in buffer composed of6×SSC, 5×Denhart's 0.5% SDS, 100 mg/mL salmon sperm DNA. Hybridizationsare carried out in the game solution upon adding 5-20×10 ⁶ cpm of³²P-labeled probe and incubated 8-48 hours at 55° C. Washing of filtersis done at 60° C. in 1×SSC, 0.1% SDS, with two exchanges after 30minutes. Other conditions for moderately high stringency screening areknown in the art. For further guidance regarding hybridizationconditions see, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al.,1989, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y.

The invention also relates to α(2-3) trans-sialidase derivatives oranalogs made by altering the α(2-3) trans-sialidase sequence bysubstitutions, additions or deletions that provide for molecules withα(2-3) trans-sialidase activity (i.e., catalyzes the transfer of sialicacid from one saccharide-containing molecule to another). Thus, theα(2-3) trans-sialidase derivatives include polypeptides containing, as aprimary amino acid sequence, all or part of the α(2-3) trans-sialidaseamino acid sequence including altered sequences in which functionallyequivalent amino acid residues are substituted for residues within thesequence resulting in a polypeptide which is functionally active (i.e.,a polypeptide possessing trans-sialidase activity). For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Conservative substitutionsfor an amino acid within the sequence may be selected from other membersof the class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Such α(2-3)trans-sialidase derivatives can be made either by chemical peptidesynthesis or by recombinant production from nucleic acid encoding theα(2-3)-trans-sialidase which have been mutated. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson etal., 1978, J. Biol. Chem 253:6551), use of TAB® linkers (Pharmacia),etc.

The trans-sialidase, or functionally active derivative (includingfragments and fusion proteins), or analog used according to the methodof the present invention can be obtained by purification from biologicaltissue or cell culture, or produced by recombinant or synthetictechniques known in the art.

Native α(2-3) trans-sialidase preparations can be obtained from avariety of sources. Standard methods for protein purification may beused to isolate and purify, or partially purify, α(2-3) trans-sialidasesfrom any source known to contain or produce the desired α(2-3)trans-sialidase, e.g., T. cruzi or T. brucai. Such standard proteinpurification techniques include, but are not limited to, chromatography(e.g., ion exchange, affinity, gel filtration/molecular exclusionchromatography and reversed phase high performance liquid chromatography(RP-HPLC)), centrifugation, differential solubility, and electrophoresis(for a review of protein purification techniques, see, Scopes, ProteinPurification; Principles and Procedure, 2nd Ed., C. R. Cantor, Editor,Springer Verlag, New York, N.Y. (1987), and Parvez et al., Progress inHPLC, Vol. 1, Science Press, (1985) Utrecht, The Netherlands). Forexample, antibodies to trans-sialidases may be generated usingtechniques known in the art, and can be used to prepare an affinitychromatography column for purifying the respective trans-sialidases bywell-known techniques (see, e.g., Hudson & May, 1986, PracticalImmunology, Blackwell Scientific Publications, Oxford, United Kingdom).

5.1.1. Recombinant Production of Trans-Sialidase

Recombinant expression techniques can be applied to obtain the α(2-3)trans-sialidases, derivatives, and analogs utilized according to themethod of the invention (see, e.g., Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d Ed.,Cold Spring Harbor, N.Y., Glover, D. M. (ed.), 1985, DNA Cloning: APractical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II). Thenucleic acid sequences of α(2-3) trans-sialidases such as, for example,those described above, are known and can be isolated using well-knowntechniqueg in the art, such as screening a genomic or cDNA library,chemical synthesis, or polymerase chain reaction (PCR). Further, giventhese known sequences, other α(2-3) trans-sialidases may be cloned usingroutine recombinant techniques known in the art, such as, for example,PCR and hybridization to the complement of the known nucleic acidsequence under highly stringent, moderately highly stringent, and lowstringency conditions, in combination with assays which select for knownbiochemical properties of the α(2-3) trans-sialidase of interest, orgenerally, to catalyze the transfer of sialic acid from a donorsaccharide-containing molecule to an acceptor saccharide-containingmolecule. Cloned α(2-3) trans-sialidase gene sequence can be modified byany of numerous strategies known in the art.

To recombinantly produce a α(2-3) trans-sialidase, derivative or analog,a nucleic acid sequence encoding the α(2-3) trans-sialidase, derivative,or analog, is operatively linked to a promoter such that the α(2-3)trans-sialidase, derivative, or analog is produced from said sequence.For example, a vector can be introduced into a cell, within which cellthe vector or a portion thereof is expressed, producing an α(2-3)trans-sialidase or a portion thereof. In a preferred embodiment, thenucleic acid is DNA if the source of RNA polymerase is DNA-directed RNApolymerase, but the nucleic acid may also be RNA if the source ofpolymerase is RNA-directed RNA polymerase or if reverse transcriptase ispresent in the cell or provided to produce DNA from the RNA. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.

A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include, but are not limited to,mammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA. The expression elements of vectors vary in their strengths andspecificities and depending on the host-vector system utilized, any oneof a number of suitable transcription and translation elements may beused.

Expression of an α(2-3) trans-sialidase, derivative, or analog may becontrolled by any promoter/enhancer element known in the art. Suchpromoters include, but are not limited to: the SV40 early promoterregion (Bernoist and Chambon, 1981, Nature 290:304-310), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., 1980, Cell 22:787-797), the HSV-1 (herpes simplex virus-1)thymidine kinase promoter (Wagner at al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:19-42); prokaryotic expressionvectors such as the β-lactamase promoter (Villa-Kamaroff et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoeret al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also “UsefulProteins from Recombinant Bacteria” in Scientific American, 1980,242:74-94; plant expression vectors comprising the nopaline synthetasepromoter region (Herrera-Estrella et al., 1983, Nature 303:209-213) orthe cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981,Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzymeribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature310:115-120); promoter elements from yeast or other fungi such as theGal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in traneggnic animals;elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumingene control region which is active in liver (Pinkert et al., 1987,Genes and Devel. 1:268-276), alpha-fetoprotein gene control region whichis active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5;1639-1648;Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene controlregion which is active in the liver (Kelsey et al., 1987, Genes andDevel. 1:161-171), beta-globin gene control region which is active inmyeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al.,1986, Cell 46:89-94; myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., 1987,Cell 49:703-712); myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, 1985, Nature 314:283-286), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., 1986, Science 234:1372-1378). Thepromoter element which is operatively linked to the nucleic acidencoding trans-sialidase, derivative or analog, can also be abacteriophage promoter with the source of the bacteriophage RNApolymerase expressed from a gene for the RNA polymerase on a separateplasmid, e.g., under the control of an inducible promoter, for example,the nucleic acid encoding trans-sialidase, derivative, or analog,operatively linked to the T7 RNA polymerase promoter with a separateplasmid encoding the T7 RNA polymerase. In a preferred embodiment of theinvention, expression of a α(2-3) trans-sialidase, derivative, or analogis controlled by a regulatory sequence of a gene expressed in mammarytissue, such as, for example, the regulatory sequence of a gene encodinga milk specific protein (see e.g., Wright et al., 1991, Biotechnology(NY) 9:830-834; Carver et al., 1993, Biotechnology (NY) 11:1263-1270);Clark et al., 1989, Biotechnology (NY) 7:487-492; Velander et al., 1992,Proc. Natl. Acad. Sci. USA, 89:12003-12007; Ebert et al., 1991,Biotechology (NY) 9:835-838).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered α(2-3) trans-sialidase,derivative or analog may be controlled. Furthermore, different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification (e.g., glycosylation,phosphorylation of proteins). Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in a bacterial systemcan be used to produce an unglycosylated core protein product.Expression in yeast will produce a glycosylated product. Expression inmammalian cells can be used to ensure “native” glycosylation of aheterologous protein. Furthermore, different vector/host expressionsystems may effect processing reactions to different extents.

The α(2-3) trans-sialidase-encoding nucleic acid sequence can be mutatedin vitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regions.Any technique for mutagenesis known in the art can be used, includingbut not limited to, in vitro site-directed mutagenesis (Hutchinson etal., 1978, J. Biol. Chem. 253:6551), use of TAB® linkers (Pharmacia),etc.

The experimentation involved in mutagenesis consists primarily ofsite-directed mutagenesis followed by phenotypic testing of the alteredgene product. Some of the more commonly employed site-directedmutagenesis protocols take advantage of vectors that can provide singlestranded as well as double stranded DNA, as needed. Generally, themutagenesis protocol with such vectors is as follows. A mutagenicprimer, i.e., a primer complementary to the sequence to be changed, butconsisting of one or a small number of altered, added, or deleted bases,is synthesized. The primer is extended in vitro by a DNA polymerase and,after some additional manipulations, the now double-stranded DNA istransfected into bacterial cells. Next, by a variety of methods, thedesired mutated DNA is identified, and the desired protein is purifiedfrom clones containing the mutated sequence. For longer sequences,additional cloning steps are often required because long inserts (longerthan 2 kilobases) are unstable in those vectors. Protocols are known toone skilled in the art and kits for site-directed mutagenesis are widelyavailable from biotechnology supply companies, for example from AmershamLife Science, Inc. (Arlington Heights, Ill.) and Stratagene CloningSystems (La Jolla, Calif.).

In specific embodiments, the α(2-3) trans-sialidase derivative or analogused according to the method of the invention is generated bysite-directed mutagenesis of the DNA encoding a non-functional α(2-3)trans-sialidase. In a specific embodiment the codon encoding for theamino acid at position 342 (relative to Try³⁴² of Genbank L26499) ismutated to encode for a tyrosine residue. In the another specificembodiment, a more active α(2-3) trans-sialidase is generated, by thesite-directed mutagenesis of DNA encoding a less active α(2-3)trans-sialidase by mutating the codon encoding as for the amino acid atposition 231 of the less active α(2-3) trans-sialidase (relative toPro²³¹ of Genbank L26499) to encode a proline residue.

In other specific embodiments, the α(2-3) trans-sialidase derivative oranalog may be expressed as a fusion, or chimeric protein product(comprising the protein, fragment, analog, or derivative joined via apeptide bond to a heterologous protein sequence (of a differentprotein)). Such a chimeric product can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acidsequences to each other by methods known in the art, in the propercoding frame, and expressing the chimeric product by methods commonlyknown in the art (see e.g., Section 5.6).

5.1.2. Chemical Synthesis of Trans-Sialidase

In addition, α(2-3) trans-sialidases, derivatives (including fragmentsand chimeric proteins), and analogs can be chemically synthesized. See,e.g., Clark-Lewis et al., 1991, Biochem. 30:3128-3135 and Merrifield,1963, J. Amer. Chem. Soc. 85:2149-2156. For example, α(2-3)trans-sialidases, derivatives and analogs can be synthesized by solidphase techniques, cleaved from the resin, and purified by preparativehigh performance liquid chromatography (e.g., see Creighton, 1983,Proteins, Structures and Molecular Principles, W. H. Freeman and Co.,N.Y., pp. 50-60). α(2-3) trans-sialidases, and derivatives and analogsthereof can also be synthesized by use of a peptide synthesizer. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins, Structures and Molecular Principles, W. H.Freeman and Co., N.Y., pp. 34-49). Furthermore, if desired, nonclassicalamino acids or chemical amino acid analogs can be introduced as asubstitution or addition into the α(2-3) trans-sialidase, derivative oranalog. Non-classical amino acids include but are not limited to theD-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, degigner amino acids such as β-methyl amino acids,Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

By way of example, but not by way of limitation, proteins (includingpeptides) of the invention can be chemically synthesized and purified asfollows: α(2-3) trans-sialidases, derivatives and analogs can besynthesized, by employing the N-α-9-fluorenylmethyloxycarbonyl or Fmocsolid phase peptide synthesis chemistry using a Rainin symphonyMultiplex Peptide Synthesizer. The standard cycle used for coupling ofan amino acid to the peptide-resin growing chain generally includes: (1)washing the peptide-resin three times for 30 seconds withN,N-dimethylformamide (DMF); (2) removing the Fmoc protective group onthe amino terminus by deprotection with 20% piperdine in DMF by twowashes for 15 minutes each, during which process mixing is effected bybubbling nitrogen through the reaction vessel for one second every 10seconds to prevent peptide-resin settling; (3) washing the peptide-resinthree times for 30 seconds with DMF; (4) coupling the amino acid to thepeptide resin by addition of equal volumes of a 250 mM solution of theFmoc derivative of the appropriate amino acid and an activator mixconsisting or 400 mM N-methylmorpholine and 250 mM(2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) in DMF; (5) allowing the solution to mix for 45 minutes; and (6)washing the peptide-resin three times for 30 seconds of DMF. This cyclecan be repeated as necessary with the appropriate amino acids insequence to produce the desired polypeptide. Exceptions to this cycleprogram are amino acid couplings predicted to be difficult by nature oftheir hydrophobicity or predicted inclusion within a helical formationduring synthesis. For these situations, the above cycle can be modifiedby repeating step 4 a second time immediately upon completion of thefirst 45 minute coupling step to “double couple” the amino acid ofinterest. Additionally, in the first coupling step in polypeptidesynthesis, the resin can be allowed to swell for more efficient couplingby increasing the time of mixing in the initial DMF washes to three 15minute washes rather than three 30 second washes. After polypeptidesynthesis, the peptide can be cleaved from the resin as follows: (1)washing the polypeptide-resin three times for seconds with DMF; (2)removing the Fmoc protective group on the amino terminus by washing twotimes for 15 minutes in 20% piperdine in DMF; (3) washing thepolypeptide-resin three times for 30 seconds with DMF; and (4) mixing acleavage cocktail consisting of 95% trifluoroacetic acid (TFA), 2.4%water, 2.4% phenol, and 0.2% triisopropysilane with thepolypeptide-resin for two hours, then filtering the peptide in thecleavage cocktail away from the resin, and precipitating the peptide outof solution by addition of two volumes of ethyl ether. To isolate thepolypeptide, the ether-peptide solution can be allowed to sit at −20° C.for 20 minutes, then centrifuged at 6,000×G for 5 minutes to pellet thepolypeptide, and the polypeptide can be washed three times with ethylether to remove residual cleavage cocktail ingredients. The finalpolypeptide product can be purified by reversed phase high pressureliquid chromatography (RP-HPLC) with the primary solvent consisting of0.1% TFA and the eluting buffer consisting of 80% acetonitrile and 0.1%TFA. The purified polypeptide can then be lyophilized to a powder.

5.2. Assays for α(2-3) Trans-sialidase Activity

The invention is based in part on the discovery that the addition ofα(2-3) trans-sialidase to a dairy source in sufficient quantities tocatalyze the transfer of sialic acids from the sialyloligosaccharidepopulation of the dairy source will favor the sialylation of lactose dueto the high concentration of lactose in dairy sources. Thus, the abilityof α(2-3) trans-sialidases, and derivatives and analogs thereof tocatalyze the transfer of sialic acid from an α(2-3)sialyloligosaccharide donor source to an oligosaccharide acceptor havinga β-galactose moiety at its non-reducing terminus is indicative of theusefulness of these proteins, derivatives, and analogs in producingsialyloligosaccharides in a dairy source or cheese processing wastestream according to the methods of the present invention.

The trans-sialidases that may be used according to the methods of theinvention encompass all protein sequences with functional α(2-3)trans-sialidase activity. The α(2-3) trans-sialidases, therefore, aredefined by catalytic activity in which the α(2-3) trans-sialidasedirects the transfer of a sialic acid from one saccharide containingmolecule (e.g., oligosaccharide, polysaccharide, glycoprotein, orglycolipid) to another. Assays for α(2-3) trans-sialidase activity arewell known in the art and may be applied according to the presentinvention, both to identify α(2-1) trang-gialidases, derivatives andanalogs demonstrating the requisite catalytic activity, and also foroptimization of reaction parameters (e.g. concentration, temperature, pHand incubation time) for incubating the α(2-3) trans-sialidase,derivative, or analog with the dairy source or cheese processing wastestream.

In one embodiment, α(2-3) trans-sialidase activity is measured using themethod described in Vandekerckhove, et al. 1992, Glycobiology 2:541-548.Briefly, α(2-3) trans-sialidase is incubated in 20 mM Hepes buffer(Sigma H-3375) at pH=7.2 in the presence of α(2-3) sialyllactose and[D-glucose-1-¹⁴C]lactose (60 mCi/mmol) (Amersham, Arlington Heights,Ill.). Reactions are stopped by the addition of 20 μl ethanol. Theresulting compounds are analyzed by thin layer chromatography (TLC) onsilica gel plates (EM Science, HPTLC Fertigplatten Kieselgel 60F254,10×10 cm) and chromatographed in ethanol-n-butanol-pyridine-water-aceticacid [100:1010:30:3 (v/v)]. Sialic acid-containing molecules arevisualized by resorcinol staining against Neu5Ac, MU-Neu5Ac, α(2-3)sialyllactose and α(2-6) sialyllactose standards.

Other assays for α(2-3) trans-sialidase activity are known in the artand may be used according to the present invention to assess for and/orto optimize α(2-3) trans-sialidase activity. Further, assays forglycosyltransferase activity known in the art may also be routinelymodified so as to test for and/or optimize α(2-3) trans-sialidaseactivity.

5.3. Enrichment of α(2-3) Sialyloligosaccharides Including α (2-3)Sialyllactose

The invention provides methods for producing sialyloligosaccharides,particularly α(2-3) sialyllactose, in a dairy source or in a cheeseprocessing waste stream.

In one embodiment, the present invention provides a method for producingsialyloligosaccharides in a dairy source. This method comprisescontacting a catalytic amount of at least one α(2-3) trans-sialidasewith a dairy source to form a dairy/trans-sialidase mixture, andincubating the dairy/trans-sialidase mixture under conditions suitablefor α(2-3) trans-sialidase activity.

In another embodiment, the present invention provides a method forproducing sialyloligosaccharides in a cheese processing waste stream.This method comprises contacting a catalytic amount of at least oneα(2-3) trans-sialidase with a cheese processing waste stream to form awaste stream/trans-sialidase mixture and incubating the wastestream/trans-sialidase mixture under conditions suitable for α(2-3)trans-sialidase activity.

In an additional embodiment of the present invention,sialyloligosaccharides are produced and recovered from a dairy source bya method comprising contacting a catalytic amount of at least one α(2-3)trans-sialidase with a dairy source to form a dairy/trans-sialidasemixture, incubating the dairy/trans-sialidase mixture under conditionssuitable for α(2-3) trans-sialidase activity, and recovering thesialyloligosaccharides from the incubated dairy/trans-sialidase mixture.

The present invention also provides a method for producingsialyloligosaccharides in a dairy source which is subsequently processedfor cheese manufacture, followed by recovery of thesialyloligosaccharides from the cheese processing waste stream. Whensialyloligosaccharides are produced in a dairy source, processed forcheese manufacture and recovered from the cheese processing waste streamby the method of the present invention, the method comprises contactinga catalytic amount of at least one α(2-3) trans-sialidase with a dairysource to form a dairy/trans-sialidase mixture, incubating thedairy/trans-sialidase mixture under conditions suitable for α(2-3)trans-sialidase activity, processing the incubated dairy/trans-sialidasemixture using any known protocol for the manufacture of cheeses, andrecovering the sialyloligosaccharides from the cheese processing wastestream derived from the incubated dairy/trans-sialidase mixture.

In another embodiment of the present invention, sialyloligosaccharidesare produced in and recovered from cheese processing waste streams by amethod comprising contacting a catalytic amount of at least one α(2-3)trans-sialidase with a cheese processing waste stream to form a wastestream/trans-sialidase mixture, incubating the wastestream/trans-sialidase mixture under conditions suitable for α(2-3)trans-sialidase activity, and recovering sialyloligosaccharides from theincubated waste stream/trans-sialidase mixture.

In each embodiment of the methods of the present invention, the α(2-3)trans-sialidases encompass molecules with enzymatic activity wherein asialic acid is transferred from one saccharide-containing molecule toanother saccharide-containing molecule. The saccharide-containingmolecules may be oligosaccharides, polysaccharides, glycoproteins orglycolipids. The α(2-3) trans-sialidases used according to the methodsof the present invention are further defined supra in Sections 3.1 and5.1.

The α(2-3) trans-sialidase used according to the methods of the presentinvention may be a purified α(2-3) trans-sialidase, derivate or analog;a partially purified α(2-3) trans-sialidase, derivative or analog; or acrude or filtered eukarytic or bacterial (e.g. E. coli) lysatecontaining α(2-3) trans-sialidase activity. Optimal enzymeconcentrations used according to the methods of the present inventionmay beg routinely determined using techniques known in the art. Inspecific embodiments, the concentration of α(2-3) trans-sialidase usedaccording to the methods of the invention is at least 0.001, 0.005,0.01, 0.05, 0.075, 0.10 or 0.4 units/ml (wherein one unit is defined asthe concentration of enzyme required to produce 1 μmolNAN-α(2-3)-Gal-β(1-4)-GlcNAc-β(1-3)-Gal-β(1-4)-Glc (LST-d)/min in astandard assay using α(2-3)-sialyllactose andGal-β(1-4)-GlcNAc-β(1-3)-Gal-β(1-4)-Glc (lacto-N-neotetraose, LNnT) assubstrates).

The dairy sources used in the methods according to the present inventioninclude, but are not limited to, milk, colostrum, a cheese processingmixture, or a composition simulating milk. As used herein, the phrasecheese processing mixture refers to a compilation of ingredients ofdairy processing at any stage during dairy processing (e.g., cheesemanufacture) other than the cheese processing waste stream. Acomposition simulating milk is a solution lacking one or more ofCMP-sialyltransferase, CMp-gynthetase and/or free sialic acid, but whichcontains at least α(2-3) sialosides to act as donors for the α(2-3)trans-sialidase, lactose and, optionally, appropriate buffering agentsto maximize the activity of the α(2-3) trans-sialidase when it is addedto the solution. Alternatively, a composition simulating milk is asolution containing at least α(2-3) sialosides to act as donors for theα(2-3) trans-sialidase and lactose, and wherein the presence of freesialic acid, CMP-sialytransferase and/or CMP-synthetase is not requiredto drive the sialylation of lactose by α(2-3) trans-sialidase. A cheeseprocessing waste stream is the portion of cheese manufacturing notretained for cheese after formation of curd. The cheese processing wastestream typically refers to the fluid drained from curd, which isfrequently discarded. A cheese processing waste stream of the presentinvention includes, but is not limited, to whole whey, demineralizedwhey permeate, the regeneration stream from demineralized whey permeate,whey permeate, crystallized lactose, spray dried lactose, whey powder,edible lactose, and lactose.

In each embodiment of the present invention, the α(2-3) trans-sialidaseis contacted with the dairy source or cheese processing waste stream andthe resulting mixture may be agitated, stirred, mixed, or subjected toany other method of combining. Whether the α(2-3) trans-sialidase isadded to colostrum, milk, a cheese processing mixture, a compositionsimulating milk, or to milk that has undergone some processing, maydictate the amount of stirring or mixing which may be required. Whilemilk is relatively fluid, processed milk, such as milk being processedfor cheese, may become quite viscous and require more agitation,stirring, mixing, or the like for efficient enzymatic activity to occur.Likewise, a cheese processing waste stream may be a viscous solution andrequire similar forms of agitation, stirring, mixing, and the like, forefficient enzymatic activity.

Conditions suitable for producing sialyloligosaccharides, particularlyα(2-3) sialyllactose, in a dairy source or cheese processing wastestream by the methods of the present invention, may be determined andoptimized by routine techniques known in the art. In one embodiment, thedairy source or cheese processing waste stream is initially chilled to2-20° C.

The optimal time to incubate the dairy/trans-sialidase mixture generatedaccording to the present invention may be routinely determined bytechniques known in the art. In specific embodiments, thedairy/trans-sialidase mixture is incubated for a period of at least 0.5,1.0, 5.0 or 10.0 hours. In a preferred embodiment, thedairy/trans-sialidase mixture is incubated for 12-30 hours. In a morepreferred embodiment, the dairy/trans-sialidase mixture is incubated for20-25 hours.

The optimal temperature to incubate the dairy/trans-sialidase mixturegenerated according to the methods of the present invention may beroutinely determined by techniques known in the art. In specificembodiments, the dairy/trans-sialidase mixture is incubated at about0-30° C. or 2-20° C. In preferred embodiments the dairy/trans-sialidasemixture is incubated at 5-15° C. or 8-12° C. In embodiments where thedairy source is a composition simulating milk, the dairy/trans-sialidasemixture may be incubated at about 0-45° C., 10-45° C., or 20-40° C.

The optimal pH to incubate the dairy/trans-sialidase mixture accordingto the present invention may be routinely determined by techniques knownin the art. In specific embodiments, the dairy/trans-sialidase mixtureis incubated at a pH of about pH 5-9, more preferably at about pH 6-8,and most preferably the pH is at about 7.

Further conditions to optimize the incubation of thedairy/trans-sialidase mixture will be apparent to those skilled in theart and are within the scope of the present invention. In a specificembodiment, the dairy/trans-sialidase mixture may be agitated, stirred,shaken, mixed, or the like, to assist the even distribution of enzymewithin the mixture.

In one embodiment of the invention, exogenous α(2-3)sialyloligosaccharides are added to the dairy/trans-sialidase mixture.The supplemented exogenous α(2-3) sialyloligosaccharides may contain asingle homogeneous α(2-3) sialyloligosaccharide population, oralternatively, may consist of a mixture of different α(2-3)sialyloligosaccharides. α(2-3) sialyloligosaccharide supplemented duringthis incubation step should be selected so as to minimize possiblenegative effects upon the taste, texture, appearance or quality of thedairy product (e.g., cheese).

Following incubation, the milk may be pasteurized by any method ofpasteurization known in the art, including, but not limited to, HTST(High Temperature, Short Time Sterilizer/Pasteurizer) at 161°F. for 18seconds and cooled to 80° F. Sialyloligosaccharides, including, but notlimited to, α(2-3) sialyllactose, may be recovered from the incubateddairy/trans-sialidase mixture or from the pasteurizeddairy/trans-sialidase mixture by the methods described infra in Section5.4. Where the dairy/trans-sialidase mixture is to be used tomanufacture cheese, the dairy/trans-sialidase mixture is collected andprocessed for making cheese. Alternatively, milk may be pasteurizedbatchwise (rise and drop of a whole batch to 160°F. is one protocolused) or by HTST pasteurizer/heat exchanger (quick rise to 160° F., holdfor 2 minutes, quick chill to 80° F.). Milk may also be sterilized byUHT (ultrahigh temperature sterilization) (quick rise to 270° F., holdfor 6 seconds, quick chill to 80° F.). Depending on the subsequentprocess, this method of sterilization may use heat exchange or cleansteam injection. In an alternative embodiment of the invention, thedairy source is processed for cheese manufacture and thesialyloligosaccharides are recovered from the cheese processing wastestream by the methods described infra in Section 5.5.

In another embodiment of the invention, at least one α(2-3)trans-sialidase is contacted with a cheese processing waste stream.

The optimal time to incubate the waste stream/trans-sialidase mixtureaccording to this embodiment may be routinely determined by techniquesknown in the art. In specific embodiments, the wastestream/trans-sialidase mixture is incubated for a period of at least0.5, 1.0, 5.0 or 10.0 hours. In a preferred embodiment, the wastestream/trans-sialidase mixture is incubated for 5-45 hours. In a morepreferred embodiment, the waste stream/trans-sialidase mixture isincubated for 10-35 hours.

The optimal temperature to incubate the waste stream/trans-sialidasemixture according to the present invention may be routinely determinedby techniques known in the art. In specific embodiments, the wastestream/trans-sialidase mixture is incubated at about at 2-40° C.,preferably 15-37° C., most preferably 22-27° C.

The optimal pH to incubate the dairy source/trans-sialidase mixtureaccording to the present invention may be routinely determined bytechniques known in the art. In specific embodiments, the wastestream/trans-sialidase mixture is incubated at a pH of about 4-9, morepreferably at about pH 6-8, and most preferably the pH is at about pH 7.

Further conditions to optimize the incubation of the wastestream/trans-sialidase mixture will be apparent to those skilled in theart and are within the scope of the present invention. In specificembodiments the waste stream/trans-sialidase mixture may be agitated,stirred, shaken, mixed, or the like, to assist the even distribution ofenzyme within the mixture.

In one embodiment of the invention, exogenous α(2-3)sialyloligosaccharides are added to dairy source/trans-sialidasemixture. The supplemented exogenous α(2-3) sialyloligosaccharides maycontain a single homogeneous α(2-3) sialyloligosaccharide population, oralternatively, may consist of a mixture of different α(2-3)sialyloligosaccharides.

Following incubation of the waste stream/trans-sialidase mixture,sialyloligosaccharides, including, but not limited toα(2-3)sialyllactose may be recovered from the incubated wastestream/trans-sialidase mixture by the methods described infra in Section5.4.

5.4. Recovery of Sialyloligosaccharides

Sialyloligosaccharides produced according to the methods of the presentinvention may be recovered from the dairy source before or duringprocessing (e.g., pasteurization, fermentation, and/or one or more ofthe other processing steps involved in the manufacture of cheese oranother dairy product). Alternatively, sialyloligosaccharides producedaccording to the methods of the present invention may be recovered afterprocessing of the dairy source (e.g. from a cheese processing wastestream). The sialyloligosaccharides produced according to the methods ofthe invention may be recovered using methods known in the art,including, but not limited to, ultrafiltration, difiltration,electrodialysis, ion exchange chromatography and phase partitionchemistry.

In specific embodiments of the invention, α(2-3) sialyloligosaccharidesproduced according to the methods of the invention, are recovered from acheese processing waste stream (i.e., any waste stream or byproductgenerated during cheese making process). Whey containing sialic acids,is a byproduct obtained when cheese or rennet casein is produced frommilks such as cow milk, goat milk, and sheep milk. For example acidwhey, is generated by separating the solids when skim milk is coagulatedto form cottage cheese. Acid whey is characterized by a high lactic acidcontent. When cheese is prepared from whole milk, the remaining liquidis sweet whey, which can be further processed by evaporation to form drywhey powder. Sweet whey can also be dried, demineralized and evaporatedto form demineralized whey permeate. Sweet whey can also be subjected toultrafiltration to generate both a whey permeate and a whey proteinconcentrate. Whey permeate can be further processed by crystallizinglactose to form both lactose and a mother liquor. The mother liquorresulting from crystallizing lactose from a whey permeate is known inthe art as “Delac.”

When α(2-3) trans-sialidase is contacted with a dairy source before orduring cheese manufacture and sialyloligosaccharides are recovered froma cheese processing waste stream, suitable cheese processing wastestreams include but are not limited to, whole whey, demineralized wheypermeate, the regeneration stream from demineralized whey permeate, wheypermeate, crystallized lactose, spray dried lactose, whey powder, ediblelactose and lactose. Preferably the aqueous mother liquor materialresulting from crystallizing lactose (i.e., Delac) is used. When α(2-3)trans-sialidase is contacted with a cheese processing waste stream andsialyloligosaccharides are thereafter recovered, suitable cheeseprocessing waste streams include colostrum, milk, milk powder, wholewhey, demineralized whey permeate, the regeneration stream fromdemineralized whey permeate, whey permeate, and whey powder.

Fluid cheese whey is typically dried so as to produce a non-hygroscopic,highly dispersable powder. Fresh fluid whey is clarified by passingthrough a desludging type clarifier. The whey is separated to removefat, then concentrated in double or triple effect evaporators to asolids content of about 62% by weight. The solids can be removed byseparation at room temperature, or more preferably, the concentratedwhey is cooled before the solids are removed.

When the cheese processing waste stream to be processed is the solidsobtained from drying whey, the solids can be first dissolved in water,preferably in an amount of about 1 to 620 g, preferably 50 to 200 g,more preferably about 100 g of solids per Liter of water. Dissolution ofthe solids obtained from drying cheese whey can be conducted at roomtemperature or at elevated temperatures to accelerate the dissolutionprocess and increase the amount of dissolved solids. Preferably,temperatures of from 20-80° C. are suitable. Alternatively, the solidscan be processed directly by extraction with a solvent.

In one embodiment of the invention, sialyloligosaccharides producedaccording to the methods of the invention are recovered from a dairysource or cheese processing waste stream by a method comprising:adjusting the pH of the dairy source or cheese processing waste streamto form an acidic mixture; contacting this acidic mixture with a cationexchanger; and concentrating and desalting the eluent. See e.g.,Shimatani et al., U.S. Pat. No. 5,270,462, the contents of which areherein incorporated by reference herein in its entirety).

In another embodiment of the invention, sialyloligosaccharides producedaccording to the methods of the invention are recovered from a dairysource or cheese processing waste stream by a method comprising:subjecting a dairy source or cheese processing waste stream toultrafiltration, fractionating at 20,000 to 500,000 Daltons at a pH of4.0 to 6.0 to form a ultrafiltrate and subjecting the resultingultrafiltrate to a second ultrafiltration, fractionating at 1,000 to10,000 Daltons at a pH of 6.0 to 8.0 under 0.2 to 2.0 MPa, to removeimpurities such as protein. See e.g., JP Kokai 01-168,693, the contentsof which are incorporated by reference in its entirety.

In another embodiment of the invention, sialyloligosaccharides producedaccording to the methods of the invention are recovered from a dairysource or cheese processing waste stream by a method comprising:desalting the dairy source or cheese processing waste stream and passingthe desalted solution through an anion exchange column. See e.g., JPKokai 59-184,197 the contents of which are herein incorporated byreference in its entirety.

Other methods which may be used during the recovery ofsialyloligosaccharides produced according to the methods of the presentinvention include ultrafiltration (see e.g., U.S. Pat. No. 4,001,198 toThomas and U.S. Pat. No. 4,202,909 to Pederson); concentration andaddition of a divalent cation (see e.g., U.S. Pat. No. 4,547,386 toChambers et al.); separation and fermentation (see e.g., U.S. Pat. No.4,617,861 to Armstrong); demineralization using an electrolytic cell(see e.g., U.S. Pat. Nos. 4,971,701 and 4,855,056 to Harju et al.);separation on a bed of strongly acidic cation exchange resin (see e.g.,U.S. Pat. No. 4,543,261 to Harmon et al.); electrodialysis or an ionexchange by a cation-exchange resin and a strongly basic anion-exchangeresin, or electrodialysis and ion exchange by the cation-exchange resinand the strongly basic anionexchange resin to desalt the permeate (seee.g., U.S. Pat. No. 5,118,516 to Shimatani). The disclosures of each ofthe references cited in this paragraph are incorporated by reference intheir entireties.

In a preferred embodiment, the sialyloligosaccharides produced accordingto the methods of the invention are recovered from a dairy source orcheese processing waste stream utilizing an anion exchange resin.According to this embodiment, the dairy source or cheese processingwaste stream is optionally pretreated to remove positively chargedmaterials using techniques known in the art (see e.g., DeWitt et al.,1986, Neth. Milk Dairy J. 40:41-56; and Ayers et al., 1986, New ZealandJ. Dairy Sci. & Tech. 21:21-35; JP Kokai 52-151200 and 63-39545 and JP2-104246 and 2-138295).

Suitable cation exchange resins may be prepared by conventionaltechniques known to those of ordinary skill in the art. For example, asuitable cation exchange resin may be produced from a mixture ofpolymerizable monofunctional and polyfunctional monomer by radicalemulsion polymerization techniques, then functionalized with acidicgroups such as carboxylic acid groups or sulfonic acid groups that existin the protonated form.

The degree of crosslinking in the cation exchange resin can be chosen,depending on the operating conditions of the cation exchange column. Ahighly crosslinked resin offers the advantage of durability and a highdegree of mechanical integrity, however suffers from a decreasedporosity and a drop off in mass-transfer. A low-crosslinked resin ismore fragile and tends to swell by absorption of mobile phase. Asuitable resin may have from 2 to 12% crosslinking, preferably 8%crosslinking.

The particle size of the cation exchange resin is selected to allow forefficient flow of the dairy source or cheese processing waste stream,while still effectively removing the positively charged materials, Asuitable particle size for a column 30×18 cm is 100-200 mesh.

Suitable cation exchange resins include but are not limited toCM-Sephadex, SP-Sephadex, CM-Sepharose, S-Sepharose, CM-Cellulose,Cellulose Phosphate, Sulfoxyethyl-Cellulose, Amberlite, Dowex-50W, DowexHCR-S, Dowex Macroporous Resin, Duolit C433, SP Trisacryl Plus-M, SPTrisacryl Plus-LS, Oxycellulose, AG 50W-X2, AG50W-X4, AG50W-X8, AG50W-X12, AG 50W-X16, AG MP-50 Resin, Bio-Rex 70. More preferablysuitable resins are DOWEX TM 50 x 8 (an aromatic sulfonic acid linked toa polystyrene crosslinked resin from Dow Chemical) and AMBERLYST TM-15,AMBERLITE TM IR-120 AND AMBERLITE TM-200 acidic resins.

The dairy source or cheese processing waste stream can be contacted withthe cation exchange resin, in any suitable manner which would allowpositively charged materials to be absorbed onto the cation exchangeresin. Preferably, the cation exchange resin is loaded onto a column,and the dairy source or cheese processing waste stream is passed throughthe column, to remove the positively charged materials. An amount ofcation exchange resin is selected to affect removal of the positivelycharged materials, and will vary greatly depending on the dairy sourceor cheese processing waste stream being treated. Typically, if a wheypermeate is being treated, the loading ratio of cheese processing wastestream to cation exchange resin may be from 5 to 20, preferably from8-15, more preferably from 9 to 12:1 v/v.

When contacting is effected in a column, the dairy source or cheeseprocessing waste stream is preferably passed at a rate of from 1 to 70cm/min, preferably from 2 to 15 cm/min, more preferably at a rate of 4.6cm/min. A suitable pressure may be selected to obtain the desired flowrate. Typically a pressure of from 0 to 100 PSIG is selected. Suitableflow rates may also be obtained by applying a negative pressure to theeluting end of the column, and collecting the eluent. A combination ofboth positive and negative pressure may also be used.

The temperature used to contact the dairy source or cheese processingwaste stream with the cation exchange resin is not particularly limited,so long as the temperature is not too high to cause decomposition of thecomponents of the dairy source or waste stream. Generally ambient roomtemperature of from 17° C. to 25° C. is used.

Alternatively, the positively charged materials can be removed by suchtechniques as electrodialysis, ultrafiltration, reverse osmosis or saltprecipitation.

After the optional treatment of the dairy source or cheese processingwaste stream to remove the positively charged materials, the dairysource or cheese processing waste stream is contacted with an anionexchange resin.

Suitable anion exchange resins may be prepared by conventionaltechniques known to those of ordinary skill in the art. For example, asuitable anion exchange resin may be produced from a mixture ofpolymerizable monofunctional and polyfunctional monomer by radicalemulsion polymerization techniques, then functionalized with stronglybasic groups such as quaternary ammonium groups.

The degree of crosslinking in the anion exchange resin can be chosen,depending on the operating conditions of the to anion exchange column. Asuitable resin may have from 2 to 12% crosslinking, preferably 8%crosslinking.

The particle size of the anion exchange resin is selected to allow forefficient flow of the dairy source or cheese processing waste stream,while still effectively removing the negatively charged materials. Asuitable particle size for a column 30×18 cm is 100-200 mesh.

Suitable anion exchange resins include but are not limited to DEAESephadex, QAE Sephadex, DEAE Sepharose, Q Sepharose, DEAE sephacel, DEAECellulose, Ecteola Cellulose, PEI Cellulose, QAE Cellulose, Amberlite,Dowex 1-X2, Dowex 1-X4, Dowex 1-X8, Dowex 2-X8, Dowex MacroporousResins, Dowex WGR-2, DEAE Trisacryl Plus-M, DEAE Trisacryl Plus-LS,Amberlite LA-2, AG 1-X2, AG 1-X4, AG 1-X8, AG 2-X8, AG MP-1 Resin, AG4-X4, AG 3-X4, Bio-Rex 5 and ALIQUAT-336 (tricaprylylmethylammoniumchloride from Henkel Corp.). More preferably suitable anion exchangeresins are DOWEX TM 1×8 (a methylbenzyl ammonium linked to a polystyrenecrosslinked resin from Dow Chemical) and AMBERLYSTE TM A-26, AMBERLITETM IRA 400. AMBERLITE TM IRA 400, AMBERLITE TM IRA 416 and AMBERLITE TMIRA 910, strongly basic resins.

The dairy source or cheese processing waste stream can be contacted withthe anion exchange resin, in any suitable manner which would allow thenegatively charged materials to be absorbed onto the anion exchangeresin. Preferably the anion exchange resin is loaded onto a column, andthe dairy source or cheese processing waste stream is passed through thecolumn, to absorb the negatively charged materials onto the resin.

An amount of anion exchange resin is selected to affect absorption ofthe negatively charged materials and will vary greatly depending on thedairy source or cheese processing waste stream being treated. Typically,when the waste stream is whey permeate, the loading ratio of cheeseprocessing waste stream to anion exchange resin is from 5 to 200,preferably from 8-15, more preferably from 9 to 12:1 v/v. Whencontacting is affected in a column, the dairy source or cheeseprocessing waste stream is preferably passed at a rate for from 1 to 70cm/min, preferably from 2 to 15 cm/min, more preferably at a rate of 4.6cm/min.

A suitable pressure may be selected to obtain the desired flow rate.Typically a pressure of from 0 to 100 PSIG is selected. Suitable flowrates may also be obtained by applying a negative pressure to theeluting end of the column, and collecting the eluent. A combination ofboth positive and negative pressure may also be used.

The temperature used to contact the dairy source or cheese processingwaste stream with the anion exchange resin is not particularly limited,so long as the temperature is not too high to cause decomposition of thecomponents of the dairy source or waste stream. The pH of the wheystream may also be adjusted in addition to the temperature. Generallyambient room temperature of from 17° to 25°0 C. and a pH of from 4 to 9is used.

Upon contacting the eluent with the anion exchange resin, the negativelycharged components of the dairy source or cheese processing waste streamare absorbed onto the anion exchange resin. The materials absorbed ontothe anion exchange resin are negatively charged materials from a dairysource or cheese processing waste stream, which includes but is notlimited to sialyloligosaccharides such as α(2-3) sialyllactose α(2-6)sialyllactose and (2-6) sialyllactosamine.

The resulting liquid, after contacting with the anion exchange resin,which contains primarily water and lactose may be dried and disposed ofas animal feed, fertilizer or as a food supplement.

The anion exchange resin is then purged of the sialyloligosaccharide byeluting with an aqueous solution of a suitable salt such as sodiumacetate, ammonium acetate, sodium chloride, sodium bicarbonate, sodiumformate, ammonium chloride or a lithium salt such as lithium acetate,lithium bicarbonate, lithium sulfate, lithium formate, lithiumperchlorate, lithium chloride and lithium bromide as an eluent. Purgingan anion exchange resin with an aqueous salt can be accomplished byconventional means known to those of ordinary skill in the art. Thesialyloligosaccharide can also be removed from the anion exchange resinwith an aqueous alkali solution, although, the concentration of theaqueous alkali must be dilute enough so as not to destroy the structureof the sialyloligosaccharide. Suitable desorbing conditions can bedetermined through routine experimentation.

When eluted with an aqueous solution of lithium salts, no desalting byreverse osmosis is necessary. The entire eluent can be concentrated anddried, then the remaining solids washed with an organic solvent. Thelithium salts are dissolved and the lithium salt of thesialyloligosaccharide remains as a solid. Specifically the lithium saltsof α(2-3) sialyllactose, α(2-6) sialyllactose and α(2-6)sialyllactosamine have been found to have very low organic solventsolubility.

The lithium salts used in the eluent should be freely soluble in water,and have a high solubility in an organic solvent. In the context of thepresent invention, a high solubility in an organic solvent is ≧1 gm oflithium salt per mL of organic solvent, preferably ≧5 gm/mL, morepreferably ≧10 gm/mL at the temperature the solids are being washed.Suitable lithium salts which have been found to be freely soluble inwater and have a high solubility in organic solvents include, lithiumacetate, lithium bicarbonate, lithium sulfate, lithium formate lithiumperchlorate, lithium chloride and lithium bromide.

The organic solvent used to wash the concentrated aluent should dissolvethe eluting lithium salt, yet have a low solvating effect on the lithiumsalt of a sialyloligosaccharide. In the context of the presentinvention, a low solvating effect on the lithium salt of asialyloligosaccharide is when the solubility of the lithium salt of thesialyloligosaccharide is ≦0.5 gm per mL of organic solvent, preferably≦0.25 gm/mL, more preferably ≦0.1 gm/mL at the temperature the solidsare being washed. Suitable solvents include but are not limited toacetone, methyl ethyl ketone, 3-pentanone, diethyl ether, t-butyl methylether, methanol, ethanol and a mixture thereof.

The organic solvent preferably contains ≦0.1% wt., more preferably≦0.01% wt. of water, most preferably the organic solvent is anhydrous.The use of an organic solvent containing high concentrations of wateir,results in dissolution of the lithium salts of thesialyloligosaccharide. The temperature of the organic solvent is notparticularly limited, however preferably the organic solvent is at roomtemperature or below, more preferably 0°-5° C.

Due to the high hygroscopicity of the lithium salts of thesialyloligosaccharide, washing of the solids are conducted underconventional conditions which are known to those of ordinary skill inthe art, to limit the absorption of atmospheric moisture. For examplesuch washing can be conducted under an inert atmosphere, in a dry box orusing a Schlenk-type apparatus.

When purging the anion exchange resin, with an eluent, a suitablepurging solution is 50 mM. The pH of the eluent is preferably adjustedto be from 4 to 9, more preferably from 5 to 6. Generally from 2 to 5,preferably 4 column volumes of purging solution are used to remove thesialyloligosaccharides from the anion exchange resin, preferablyperformed at ambient temperature. Preferably, lithium acetate is used topurge the anion exchange resin of the sialyloligosaccharides.

The sodium salt of the sialyloligosaccharide can be obtained byconventional ion-exchange techniques, known to those of ordinary Skillin the art.

When an eluent other than a lithium salt is used to remove thesialyloligosaccharides from the anion exchange resin, the eluentcontaining the sialyloligosaccharides and the salt, can be concentratedand desalted, such as by subjecting the eluent to reverse osmosis toremove the salt from the sialyloligosaccharide. Reverse osmosis can beconducted through a membrane with a 100 to 700 Dalton molecular weightcut off, preferably a 400 Dalton cut-off.

Reverse osmosis is preferably conducted at a pressure of from 300-1,600psi, more preferably from 400-600 psi, even more preferably at apressure of 450 psi.

After the salts have been removed by reverse osmosis, the resultingmaterial can be concentrated to provide a solid material containinggialyloligosaccharides such as α(2-3) sialyllactose and α(2-6)sialyllactose, which can be recrystallized from a mixture of water andorganic solvents.

Preferably precipitation solvents are selected from the group ofethanol, acetone, methanol, isopropanol, diethyl ether, t-butylmethylether, ethyl acetate, hexane, tetrahydrofuran and water.

In addition, the eluent, from the aniqn exchange column, which containsa mixture of sialyloligosaccharides which includes α(2-3) sialyllactose,α(2-6) sialyllactose and α(2-6) sialyllactosamine, can be subjected toseparation of the sialyloligosaccharides contained therein, by columnchromatography on a DOWEX 1×2 anion exchange resin, at pH 4 to 6 using abuffer a suitable salt such as sodium acetate, ammonium acetate or alithium salt such as lithium acetate, lithium perchlorate, lithiumchloride and lithium bromide as an eluent. A solution of lithium acetateis preferred.

Suitable anion exchange resins may be prepared by conventionaltechniques known to those of ordinary skill in the art as previouslydescribed.

The degree of crosslinking in the anion exchange reain can be chosen,depending on the operating conditions of the anion exchange column. Asuitable resin may have from 2 to 12% crosglinking, preferably 2%crosslinking.

The particle size of the anion exchange resin is selected to allow forefficient flow of the dairy source or cheese processing waste stream,while still effectively affecting chromatographic separation of thenegatively charged materials. A suitable particle size for a column20×100 cm is 200-400 mesh.

Suitable anion exchange resins include but are not limited to DEAESephadex, QAE Sephadex, DEAE Sepharose, Q Sepharose, DEAE Sephacel, DEAECellulose, Ecteola Cellulose, PEI Cellulose, QAE Cellulose, Amberlite,Dowex 1-X2, Doupex 1-X4, Dowex 1-X8, Dowex 2-X8, Dowex MacroporousResins, Dowex WGR-2, DEAE Trisacryl Plus-M, DEAE Trisacryl Plus-LS,Amberlite LA-2, AG 1-X2, AG 1-X4, AG 1-X8 AG 2-X8, AG MP-1 Resin, AG4-X4, AG 3-X4, Bio-Rex 5 and ALIQUAT-336 (tricaprylylmethylammoniumchloride from Henkel Corp.). Preferred resins are DOWEX 1×2 (atri-methylbenzyl ammonium linked to a polystyrene crosslinked resin fromDow Chemical) and AMBERLYST and AMBERLYTE basic resins.

The mixture of sialyloligosaccharides to be separated are subjected tocolumn chromatography on an anion exchange resin. An amount of anionexchange resin is selected to affect separation of the differentsialyloligosaccharides. Typically the loading ratio ofsialyloligosaccharide to anion exchange resin is from 0.1 to 5,preferably from 0.2 to 4, more preferably 1 grams of material per literof resin at a loading concentration of from 0 to 10 mM of salt. Thechromatography is conducted at a rate of from 1 to 20 cm/h, preferably4.6 cm/h superficial velocity. A suitable pressure may be selected toobtain the desired flow rate. Typically a pressure of from 0 to 22 PSIGis selected. Suitable flow rates may also be obtained by applying anegative pressure to the eluting end of the column, and collecting theeluent. A combination of both positive and negative pressure may also beused.

Any temperature may be used to contact the dairy source or cheeseprocessing waste stream with the anion exchange resin, so long as thetemperature is not too high to cause decomposition of the components ofthe sialyloligosaccharides. Generally ambient room temperature of from17° to 25° C. is used.

When the buffer eluent is a lithium salt, the individualsialyloligosaccharides can be isolated by concentrating the eluent toform a solid and washing the lithium salts away with an organic solvent.Isolation of the lithium salt of a sialyloligosaccharide from a lithiumsalt eluent is as previously described.

The sodium salt of the sialyloligosaccharide can be obtained byconventional ion-exchange techniques, known to those of ordinary skillin the art.

When the buffer eluent is not a lithium salt, the individualsialyloligosaccharides can be isolated by reverse osmosis techniques.

According to another embodiment of the present invention, a dairy sourceor cheese processing waste stream can be treated without using anion-exchange column and without using reverse osmosis.

According to this embodiment, a dairy source or cheese processing wastestream is contacted with a solvent, wherein sialyloligosaccharides areextracted.

The sialyloligosacoharides which are extracted include but are notlimited to α(2-3) sialyllactose, α(2-6) sialyllactose and α(2-6)sialyllactosamine.

A dairy source or cheese processing waste stream can be contacted with asolvent in any suitable manner to effectively extract, bysolubilization, sialyloligosaccharides.

For example solid lactose, in powder form can be packed into a column,and a solvent passed through the packed column. As the solvent passesthrough the column, the sialyloligosaccharides are extracted from thesolid lactose. To improve the solubilization of sialyloligosaccharide,the solvent can be recirculated through the column, until an equilibriumconcentration of sialyloligosaccharide is obtained in the solvent.

To improve the solubilization of sialyloligosaccharide, the solvent canbe recirculated at elevated temperature, below the thermal decompositionpoint of the sialyloligosaccharides, preferably from 27° C. to 80° C.,more preferably from 60° C. to 75° C., at ambient pressure.

A dairy source or cheese processing waste stream, can also be contactedwith a solvent, as a slurry or suspension of the dairy source or cheeseprocessing waste stream in the solvent. The dairy source or cheeseprocessing waste stream is mixed with the solvent, preferably in a 1:4v/v ratio, more preferably 1:3 v/v. The slurry or suspension is thenstirred until the sialyloligosaccharides are solubilized in the solvent.

The ratio of dairy source or cheese processing waste stream to solventis selected so as to maximize the amount of recoveredsialyloligosaccharide and minimize the amount of solvent used. Due tothe high solubility of sialyloligosaccharides in the solvent chosen, theamount of solvent is typically much less than the volume of dairy sourceor cheese processing waste stream. Accordingly when lactose is beingprocessed, it is not necessary for the lactose to be completelydissolved.

The suspension can be stirred at any temperature, below the thermaldecomposition point of the sialyloligosaccharides, preferably from 4° C.to 80° C. more preferably from 4°-27° C., at ambient pressure.

Suitable solvent systems are, water, C[1-5] alcohols, such as methanol,ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec-butanol,tert-butanol, tert-amyl alcohol and iso-amyl alcohol and a mixturethereof. The amount of water in the C[1-5] alcohol solvent system willvary depending on the alcohol used. Preferably the solvent contains from0-75% water (v/v), more preferably from 20-70% water (v/v), morepreferably from 44-66% water. A particularly preferred solvent system isan aqueous ethanol solvent containing from 44-66% water.

When elevated temperature is used, it is preferred to remove the solventfrom the column, slurry or suspension after the maximum concentration ofsialyloligosaccharide is reached, followed, by cooling of the separatedsolvent. Upon cooling of the separated solvent, solubilized lactose willcrystallize out and can be removed from the solvent containing thesialyloligosaccharide, by conventional means such as filtration,centrifugation and decanting.

An aqueous solution of lactose such as the mother liquor obtained bycrystallizing lactose, can also be treated with a solvent at elevatedtemperature, preferably from 60° to 75° C., more preferably from 68° to72° C., followed by cooling and precipitation of the lactose fromsolution. separation of the precipitated lactose from the solvent andconcentration of the solvent provides the sialyloligosaccharide.

The aqueous solution of lactose and the solvent are mixed in a ratio ofabout 1:3 v/v, preferably 1:2 v/v more preferably 1:1 v/v. A suitablesolvent for treating an aqueous solution of lactose is a C[1-5] alcohol.

The separated solvent, or column eluent can be concentrated to yieldhigh purity sialyloligosaccharide. This material can be further purifiedby recrystallization from aqueous ethanol and a suitable organicsolvent, to remove lactose impurity.

In another embodiment to the column, slurry or suspension treatmenttechnique, a portion of the extraction solvent can be removed and passedthrough an anion exchange column and the solvent returned to the system.In this fashion, the sialyloligosaccharide can be concentrated on theanion exchange column. The solvent to be passed through the anionexchange resin can be removed continuously or batch wise.

Once the anion exchange column has been saturated withsialyloligosaccharide, the column can be removed from the system andpurged to obtain sialyloligosaccharide. A suitable purging solution is120 mM LiOAc. Generally from 2 to 5, preferably 4 column volumes ofpurging solution are used to remove the negatively charged materialsfrom the anion exchange resin, performed at ambient temperature.Suitable anion exchange resins, contacting conditions and purgingconditions have been previously described above.

Sialyloligosaccharides may also be extracted from whey waste streamsusing supercritical CO₂ extraction techniques, in a method analogous tothe methods used to extract caffeine from coffee beans. A technique forthe extraction of caffeine from coffee beans using moist supercriticalCO₂ is described in U.S. Pat. Nos. 3,806,619 and 4,260,639. In general,the supercritical CO₂ extraction method comprises contacting lactose oran aqueous solution of lactose with supercritical CO₂, under conditionsto effect solubilization of sialyloligosaccharides by the supercriticalCO₂. The supercritical CO₂, containing sialyloligosaccharides isseparated from the lactose or aqueous solution of lactose, then the CO₂is removed by evaporation, leaving behind the extractedsialyloligosaccharides.

Whey containing sialic acids, is a byproduct obtained when cheese orrennet casein is produced from milks such as cow milk, goat milk andsheep milk. Due to the fat in dairy sources and the small amount of curdor fat often remains in milk whey, it is preferable that the fat contentof these compositions generated according to the method of the inventionbe previously removed by a cream separator or clarifier. In order formilk whey proteins such as beta -lactoglobulin to be efficientlyadsorbed to a cation exchanger, the dairy source or whey may bepreviously concentrated with an ultrafiltration device. Further, thedairy source or whey may be previously desalted with an electricdializer and/or an ion exchange resin.

The dairy source or whey is adjusted to a pH of 2-5 before it issubjected to the cation exchanger. As materials for adjusting the pH,any kind of materials may be used. For example, they include an acidsuch as hydrochloric acid, sulfuric acid, acetic acid, tactic acid andcitric acid. Alternatively, acidified whey which has been desalted withthe resin to have a pH of about 1-4, may be used for adjusting the pH,in order that the whey contains a high content of sialic acids. In thedairy source or whey which has been adjusted to a pH of 2-5, sialicacids are negatively, charged, while most part of dairy source or wheyprotein is positively charged. When this dairy source or whey iscontacted with the cation exchanger, dairy source or whey protein isselectively adsorbed to the cation exchanger and, as a result, sialicacids are selectively recovered as an exchanger-passed solution. If thepH of the dairy source or whey is higher than 5, sialic acids and mostpart of dairy source or whey protein are negatively charged. Therefore,the separation is not efficient, although these two can be separatedwith an anion exchanger utilizing difference in adsorption. If the pH ofthe dairy source or whey is lower than 2, sialic acids decompose andtherefore the process is not practical.

The cation exchanger-passed solution obtained according to thisembodiment may optionally be concentrated, desalted and/or dried usingtechniques known in the art. In addition, a mother liquor obtained afterthe exchanger-passed solution is concentrated and then crystallized toremove lactose may be used as a material having a high content of sialicacids. The concentration may be made by an evaporator. Thecrystallization may be made by cooling or by addition of a seed crystal.

In order to obtain a much higher sialic acids content composition, it ispreferable that the pH of the exchanger-passed solution and/or itsmother liquor be adjusted before they are concentrated and/or desalted.The concentration may be made by evaporation or by ultrafiltration. Thedesalting may be made by electric dialysis, ion exchange,ultrafiltration or diafiltration. The diafiltration is a technique forfurther increasing the protein content, wherein a liquid, which has beenconcentrated to some extent, is ultrafiltrated while simultaneouslywater is added thereto and a passing solution is withdrawn. When theexchanger-passed solution and/or its mother liquor is adjusted to a pHof 4 or higher, the concentration may be made by ultrafiltration usingan ultrafiltration membrane having a cutoff molecular weight of 2,000approximately equal to 50,000 Dalton. The concentration may be also madeby the ultrafiltration using an ultrafiltration membrane having a cutoffmolecular weight of 10,000 at a pH of 4 or lower. In other words,kappa-casein glycomacropeptide (GMP) as a sialic acid is present as amonomer at a pH of 4 or lower, while it associates into a multimonomerat a pH of above 4. As materials for adjusting the pH, any kind ofmaterials may be used. They include alkalis such as sodium hydroxide,potassium hydroxide, calcium hydroxide, potassium carbonate, sodiumcitrate, etc.

The concentrate thus obtained is a composition having a high content ofsialic acids such as GMP. Incidentally, alpha-lactalbumin, which isusually contained in milk whey together with sialic acids, may beseparated from sialic acids, for example, by ultrafiltering theexchanger-passed solution or its mother liquor at a pH of 4 or higherusing an, ultrafiltration membrane having a cutoff molecular weight of2,000 to 50,000 Dalton.

5.5. Transgenic Mammals Producing Milk Enriched For α(2-3) Sialyllactose

The α(2-3) sialyllactose content in milk may also be enriched byexpressing α(2-3) trans-sialidase, derivatives, and analogs (see Section5.1) in transgenic mammals. In one embodiment, transgenic mammals of theinvention comprise an α(2-3) trans-sialidase encoding sequence that hasbeen operably linked to a regulatory sequence of a gene expressed inmammary tissue. Similarly, the invention provides for methods forenriching for α(2-3) sialyllactose in milk comprising the steps ofintroducing a transgene comprising an α(2-3) trans-sialidase encodingsequence operably linked to a regulatory sequence of a gene expressed inmammary tissue into the germline of a mammal to produce a transgenicmammal; selecting a transgenic mammal demonstrating α(2-3)trans-sialidase activity; and obtaining milk from the selectedtransgenic mammal.

The α(2-3) trans-sialidase transgenes introduced into the transgenicanimals of the invention comprise nucleotide sequences encoding α(2-3)trans-sialidase, derivatives or analogs (as described supra in Section5.1) operably linked to regulatory sequences (i.e., inducible andnon-inducible promoters, enhancers, operators and other elements whichdrive and/or regulate expression) of a gene expressed in mammary tissue.The nucleotide coding sequence used to produce the transgenic animals ofthe invention may be regulated by any suitable regulatory sequences, butpreferred are mammalian milk protein promoter and/or regulatorynucleotide sequences. Regulatory sequences from milk-specific proteingenes which may be used to drive expression of the target sequenceinclude, but are not limited to, promoters derived from: whey acidicprotein, β-lactoglobulin, α-lactalbumin, αsl-casein, and β-casein. Seee.g., Colman, A., 1996, Am. J. Clin. Nutr. 63:639S-645S (citingHoudebine, 1994, J. Biotechnol. 43:269-87).

Many nucleotide sequences of regulatory sequences from genes expressedin mammary tissue are known (see e.g., Houdebine, 1994, J. Biotechnol.43:269-87). Alternatively, regulatory sequences contained in genomicnucleotide sequences of genes known to be expressed in mammary tissuemay be identified using techniques known in the art. For example, thegenomic nucleotide sequences located upstream of the coding sequence ofthe gene expressed in mammary tissue can be cloned adjacent to areporter gene, such as, for example, a chloramphenicol acetyltransferase (CAT) gene. The genomic sequence/reporter gene construct isthen introduced into a mammal using techniques known in the art (Seee.g., Section 5.5.1) and the presence of regulatory sequences in thegenomic sequence/reporter gene construct is indicated by reporter geneactivity, which is assayed using techniques known in the art. To moreprecisely define the regulatory elements, deletion mutants can begenerated and tested for reporter gene activity.

The regulatory sequences of the α(2-3) trans-sialidase transgene mayinclude the entire, or any portion of, the promoters, enhancers or theircorresponding genes. For example, the α(2-3) trans-sialidase/regulatorysequence transgene construct of the invention may comprise thenucleotide coding sequence for the entire mammalian milk protein, or anyportion thereof, fused in the correct coding frame to the α(2-3)trans-sialidase encoding nucleotide sequence. The expression of thesechimeric constructs may be regulated by the regulatory sequence of themammalian milk gene component of the chimeric or alternatively, by theregulatory sequence of another gene that is expressed in mammary tissue.

Additionally, the nucleotide regulatory sequences of the α(2-3)trans-sialidase transgene gene constructs, include but are not limitedto, the entire, or any portion of the endogenous milk protein promoterof the founder animal into which the α(2-3) trans-sialidase gene isbeing introduced.

Regulatory nucleotide sequences may be obtained from mammalian milkprotein genomic DNA using techniques known in the art, including, butnot limited to, PCR and hybridization screening of genomic libraries, asfurther described in Section 5.1. For a review of techniques which maybe used, see e.g., Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, NY).These techniques may additionally be applied to generate the α(2-3)trans-sialidase/regulatory transgene of the invention and to engineerchimeric gene constructs that utilize regulatory sequences other thanthe mammalian milk protein regulatory sequences. Additionally, methodswhich have been applied to construct transgenes that have successfullyexpressed proteins in the milk of transgenic mammals may routinely bemodified to generate the α(2-3) trans-sialidase transgenic mammals ofthe invention. See e.g., Wright et al., 1991, Biotechnology (NY)9:830-834; Carver et al., 1993, Biotechnology (NY) 11:1263-1270; Clarket al., 1989, Biotechnology (NY) 7:487-492); Velander et al., 1992,Proc. Natl. Acad. Sci. USA, 89:12003-12007; and Ebert et al., 1991,Biotechology (NY) 9:835-838, the contents of each of which isincorporated by reference herein in its entirety.

5.5.1. Production of Tranagenic Animals

Mammals of any species, including but not limited to, sheep, goats, pigsand cows and non-human primates, e.g., baboons, monkeys, andchimpanzees, may be used to generate α(2-3) trans-sialidase transgenicanimals of the invention. Any technique known in the art may be used tointroduce the transgene into animals to produce the founder lines oftransgenic animals. Such techniques include, but are not limited to,pronuclear microinjection (Paterson et al., 1994, Appl. Microbiol.Biotechnol. 40:691-698; Carver et al., 1993, Biotechnology (NY)11:1263-1270; Wright et al. 1991 Biotechnology (NY) 9:830-834; and Hoppeet al., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated genetransfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad.Sci., USA 82, 6148-6152), blastocysts or embryos; gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); introducing nucleic acid constructs into embryonicpleuripotent stem cells and transferring the stem cells back into theblastocyst; and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, “Transgenic Animals,” Intl. Rev. Cytol. 115, 171-229, which isincorporated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining a trans-sialidase gene, for example, nuclear transfer intoenucleated oocytes of nuclei from cultured embryonic, fetal, or adultcells induced to quiescence (Campell et al., 1996, Nature 380:64-66;Wilmut et al., 1997, Nature 385:810-813).

In addition, α(2-3) trans-sialidase transgene expression may beaccomplished by removing mammary secretory epithelium from the animals,transfecting the epithelial cells with a transqenic construct, andreintroducing the transfected epithelial cells into the animal duringthe prepartum period so that the target gene is expressed in thesubsequent lactation period. See e.g., Bremel et al., 1989, J. DairySci. 72:2826-2833. While this method has the disadvantage of providingtransient expression of α(2-3) sialyllactose, as the mammary secretoryepithelium is sloughed off during the drying off period, this technologyprovides a method by which to accomplish the goal of enriching α(2-3)sialyllactose concentrations in milk without the significant timeinvestment of creating transgenic animals.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines.

The present invention provides for transgenic animals that carry thetranagene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems.

5.5.2 Screening of Transgenic Animals

The transgenic animals that are produced in accordance with theprocedures detailed in Section 5.5.1 are preferably screened andevaluated to select those animals which may be used as suitableproducers of milk containing enriched concentrations of α(2-3)sialyllactose when compared to non-transgenic animals.

Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR) and the like.

The transgenic animals that express α(2-3) trans-sialidase protein(detected immunocytochemically, using antibodies directed against α(2-3)trans-sialidase) at easily detectable levels may serve as suitableproducers of milk containing enriched concentrations of α(2-3)sialyllactose.

5.5.3 α(2-3) Sialyllactose Enrichment in the Milk of Transgenic Mammals

The invention provides for a method for enriching for α(2-3)sialyllactose in milk comprising the steps of introducing a transgenecomprising an α(2-3) trans-sialidase encoding sequence operably linkedto a regulatory sequence of a gene expressed in mammary tissue into thegermline of a mammal to produce a transgenic mammal; selecting atransgenic mammal demonstrating α(2-3) trans-sialidase activity; andobtaining milk from the selected transgenic mammal. The α(2-3)sialyllactose produced according to this invention is also encompassedby the invention.

α(2-3) sialyllactose enriched according to the method of the inventionmay be recovered using techniques known in the art as well as thosedescribed infra (see Section 5.4).

In specific embodiments, the α(2-3) sialyllactose is recovered from themilk of the selected transgenic mammal prior to, during, or afterprocessing of the milk. In a preferred embodiment the α(2-3)sialyllactose is recovered from the milk of the selected transgenicmammal after the milk has been subjected to processing (e.g., a cheeseprocessing waste stream derived from this milk). The invention isfurther illustrated by reference to the following examples. It will beapparent to those of skill in the art that many modifications, both tomaterials and methods, may be practiced without departing from thepurpose and scope of this invention.

5.6 Example: Isolation and Cloning of a DNA Sequence Encoding α(2-3)Trans-sialidase Activity

In this Example, a chimeric DNA sequence was cloned using the polymerasechain reaction (PCR) and Trypanosoma cruz α(2-3) trans-sialidase DNA astemplate. Site directed mutagenesis was applied to alter this sequenceto encode a tyrosine at position 342 in place of the histidine initiallyencoded at this position. The mutated sequence was cloned into a pGEXexpression plasmid, transformed into a host cell, and expressed as aglutathione-S-transferase fusion protein having α(2-3) trans-sialidaseactivity.

Two sets of oligonucleotide primers (e.g., PCR Primer Set #1 and PCRPrimer Set #2) were synthesized for use in PCR to enzymatically amplifyan α(2-3) trans-sialidase encoding sequence from T. cruzi genomic DNA.

PCR Primer Set #1 was designed to amplify a region of the nucleic acidsequence that encodes the amino-terminal region of the T. cruzitrans-sialidase that maintains the active domain for α(2-3)trans-sialidase activity. In order to subsequently directionally clonethe amplified fragment, the 5′-primer was designed to include arecognition sequence for an a unique restriction endonuclease (i.e., XbaI) at the 5′ end of the amplified fragment. The sequences of 5′ primerand 3′ primer of Set #1 were (SEQ ID NO:5 and SEQ ID NO:6):

ts1xba-5′: 5′-TTTTCTAGAATGCTGGCACCCGGATCGAGC-3′

ts1-3′: 5′-CTGTGCGACAAAAAGCCAACAAGACCAACC-3′

PCR Primer Set #2 was designed to amplify a region of the nucleic acidsequence that encodes the carboxyl-terminal region of the T. cruziα(2-3) trans-sialidase and which overlapped the fragment amplified byPCR Primer Set #1. The 3′-primer was designed to include a recognitionsequence for an another unique restriction endonuclease (i.e., Xho I) atthe 3′ end of the amplified fragment. The sequences of 5′ primer and 3′primer of Set #2 were (SEQ ID NO:7 and SEQ ID NO:8):

ts2-5′: 5′-ACTGAACCTCTGGCTGACGGATAACCAGC-3′

ts2xho-3′:5′-TTTCTCGAGTCAGGCACTCGTGTCGCTGCT-3′

PCR techniques known in the art were used to amplify DNA fragmentsgenerated using each of these primer sets and T. cruzi genomic DNA astemplate.

The DNA fragments generated from the PCR of the primers of Set #1 andthe primers of Set #2 were joined to generate the full-length fragmentof the α(2-3) trans-sialidase using the following procedure. The DNAfragment generated from the PCR of the primers of Set #1 was digestedwith the restriction endonucleases XbaI and PstI. The DNA fragmentgenerated from the PCR of the primers of Set #2 was digested with XhoIand PstI. The PstI site was contained in a region that both PCR productshad in common. The digestion of the PCR products generated “sticky ends”on the products. The XbaI site at the 5′ end of the DNA fragmentgenerated from the PCR of the primers of Set #1 and the XhoI site at the3′ end of the DNA fragment generated from the PCR of the primers of Set#2 were designed to be used in the directional cloning of the entiresequence into the appropriate expression plasmid. Both PCR products werethen ligated together into Xba I/Xho I-digested pGEX (Pharmacia,Piscataway, N.J.) plasmid which contains the same restrictionendonuclease sites in its polylinker region. The α(2-3) trans-sialidasenucleotide sequence was directionally cloned in-frame of the pGEX fusiongene, glutathione-S-transferase. The trans-sialidase pGEX construct wasthen transformed into host cells using techniques known in the art.

DNA Sequence analysis revealed that the procedure described in thisexample resulted in the cloning of an α(2-3) trans-sialidase whichcontained a Tyr³⁴²→His mutation and was thus inactive (hereinafter, thisclone will be referred to as “pGEX-TS/His”). Therefore, a site directedmutagenesis protocol was followed which changed His³⁴²→Tyr.

Site-directed mutagenesis was accomplished using the following method. Aset of oligonucleotide primers (“mut-5′” and “mut-3′”) were designed tomutate His³⁴² to Tyr³⁴² in order to generate an active trans-sialidase.The sequences of mut-5′ and mut-3′ were SEQ ID NO:9 and SEQ ID NO:10):

mut-5′: 5′-GGGCAAGTATCCATTGGTGATGAAAATTCCGCCTACAGCT-3′

mut-3′: 5′-TACAGCTTATCATCCTTGTACAGGACGGAGCTGTAGGCGG-3′

The mut-5′ and mut-3′ primers were used in conjunction with the PCRprimers from PCR Primer Sets #1 and #2 to amplify overlapping DNAfragments encoding the α(2-3) trans-sialidase using pGEX-TS/His as atemplate. The primers were designed to amplify two fragments thatoverlapped by 65 nucleotides and to include PCR-directed mutations ofthe His³⁴² codon which would ultimately encode a Try³⁴². The newoverlapping fragments were gel-purified, and used in a PCR reaction asboth primer and template. That is, the two fragments were mixedtogether, heat-denatured and allowed to re-anneal in a PCR primer-freePCR reaction (which allows annealed fragments to be end-filled). The5′-end primer from PCR Primer Set #1 and the 3′-end primer from PCRPrimer Set #2 were then added to the mixture to amplify the full-length,mutated trans-sialidase-encoding fragment. The new fragment was thenligated into pCEX as described above and used to transform E. coli BL21cells (hereinafter, this clone will be referred to as “pGEX-TS/Tyr”).

E. coil bearing pGEX-TS/Tyr were expressed and assayed for α(2-3)trans-sialidase activity using techniques known in the art. Clonesexpressing α(2-3) trans-sialidase activity were isolated and utilized inthe lysate preparations utilized to generate the data presented in FIGS.5-9 and Sections 5.7 and 5.8.

5.6.1 Preparation of T. cruzi α(2-3) trans-sialidase lysates

A single colony of E. coli BL21 cells carrying pGEX-TS/Tyr wasinoculated into 2 ml of LB medium (tryptone, yeast. extract, NaCl)containing an appropriate antibiotic and the bacterial culture wasincubated at 37° C. in a shaking incubator overnight. The next day, oneliter of LB medium containing the appropriate antibiotic was inoculatedwith 50 μl of the overnight bacterial culture, and the culture wasincubated at 37° C. in a shaking incubator until the culture'sO.D.₆₀₀=1.0. The bacteria in the 1 L culture was induced to express theα(2-3) trans-sialidase by the addition of isopropylthio-β-D-galactoside(IPTG) to a final concentration of 100 μM. The induced 1 L culture wasthen placed in a shaking incubator at 20° C. overnight.

The next day, the bacteria cells were collected by centrifugation, and abacterial lysate was prepared using an APV Gaulin homogenizer. Thehomogenizer uses high pressure (10,000 psi) to lyse the cells.Alternatively, a DynoMill, french press, N₂ cavitation, douncer, freezethawing or similar tool may be used.

Alternatively, the α(2-3) trans-sialidase enzyme may be expressed usingother methods known in the art, including, but not limited to, secretoryexpression in E. coli, expression in fungi and expression in insectcells.

5.7 EXAMPLE: ENRICHMENT AND ISOLATION OF α(2-3) SIALYLLACTOSE IN MILKPRIOR TO CHEESE MANUFACTURING

In this Example, the addition of T. cruzi α(2-3) trans-sialidase to rawmilk prior to the use of this milk in the manufacture of a rennetcheese, is demonstrated to result in an enrichment of α(2-3)sialyllactose.

5.7.1 Materials and Methods

Sixty-seven gallons of fresh, raw milk were collected in 7 milk cans andchilled to 37° F. Twenty thousand units of α(2-3) trans-sialidase wereadded to each milk can. One unit of α(2-3) trans-sialidase=1 μmolNAN-α(2-3)-Gal-β(1-4)-GlcNAc-β(1-3)-Gal-β(1-4)-Glc (LST-d) produced/minin the standard assay using α(2-1) sialyllactose andGal-β(1-4)-GlcNAc-β(1-3)-Gal-β(1-4)-Glc (lacto-N-neotetraose, LNnT) assubstrates. In this particular Example, a filtered/frozen/thawed E. colilysate containing 400 units/ml α(2-3) trans-sialidase was used. The milkcontaining the α(2-3) trans-sialidase was incubated at 50° F. for 12hours. Samples were collected from 2 of the milk cans at 0, 1, 2, 3, and11 hours of the incubation and analyzed for α(2-3) sialyllactose.

After the 12 hour incubation, the milk was pasteurized by continuousHTST at 161° F. for 18 seconds and cooled to 80° F. according tostandard procedure. See Kosikowski, Frank V.; 1977, Cheese and FermentedFoods, 2nd edition, Edwards Brothers Publishing, Ann Arbor, Mich. Themilk was collected in a “double circle” vat and processed for whitecheddar cheese according to standard procedure.

5.7.1.1 Cheese Making Protocol

At Time=0, ten grams of a 33 g freeze-dried lactic acid culture (EZALEZ100 5000#, Lot No. 93053A, Rhone Polenc) were added to the pasteurizedmilk and the temperature was raised to 88° F. Three ounces of 0.02%CaCl₂ (Rhone Polenc) were added. The culture was stirred constantly at12 rpm and the temperature was held constant at 88° F. for 1 hour. Theacidity of the solution was 0.17.

At Time=1 hour and 15 minutes, milk coagulator was added. Two ounces ofa 50,000 MCU/ml Chymax solution were added to the 480# of fermented milk(3 oz of the Chymax solution are recommended/1000# (the Pfizer brandcontains chymosin, NaCl and propylene glycol). The solution was mixed,for 30 seconds and then allowed to set for30 minutes. During this stage,the milk was observed to have coagulated, forming a yogurt/sourcream-like consistency anid the titratable acidity dropped to 0.10.

At Time=1 hour and 45 min, the curd was cut into 1 cm cubes using twoknives over a period of 10 minutes.

At Time=1 hour and 55 minutes, the temperature was raised 2° F. every 5minutes until the temperature reached 102° F. The temperature was thenheld at 102° F. with constant stirring. After 30 minutes of cooking, thestir paddles were replaced with stirring rakes and the whey was allowedto drain. Eight liters of whey were collected for analysis. The pH beganto drop from the current 6.5 level, and the titratable acidity began torise from 0.10.

At Time=6 hour and 20 minutes, the pH of the whey had dropped to 5.85.The acidity had risen to 0.285 and the pH of the whey had dropped to5.85. While a lower pH and higher acidity (up to 0.6) are desired, it isbelieved that the use of an older freeze-dried lactic acid culture wasthe reason for the higher pH and lower acidity levels observed in thisexample. The curd was salted (1.25# of salt into 44# of curd), stirredand then pressed overnight at room temperature. The curd was stored at50° F. for 6 months.

5.7.1.2 Sample Dry Weight Analysis

Five ml of either the milk or whey samples were placed in pre-weighedaluminum weigh boats and placed in an 85° C. vacuum oven (<3 mmHg)overnight. The samples were then weighed and returned to the oven for anadditional 2 hours. The weighing process was repeated every 2 hoursuntil 2 consecutive, consistent readings were obtained. The net weightof the dried sample was expressed in terms of % weight per volume ofsample. The results are shown in Table 1.

5.7.1.3 α(2-3) Trans-sialidase Reaction

The milk and whey samples were frozen immediately upon collection. Foranalysis, frozen samples were thawed quickly, boiled to coagulate theremaining protein and centrifuged at 10,000×g in a microcentrifuge for10 minutes. The supernatant was collected and filtered through a 10,000MW filter to separate the α(2-3) sialyllactose from the remaining highermolecular weight compounds as a preparation for high performance liquidchromatography (HPLC). The amount of α(2-3) sialyllactose in the milksamples was quantified by HPLC and the values were expressed in terms oftotal dissolved solids.

5.7.2 Results

As demonstrated by the data presented in Table 1, the α(2-3)trans-sialidase treatment resulted in a significant increase in a α(2-3)sialyllactose within the first hour of inoculation. This level ofactivity most probably was maintained throughout the entire 11 hourincubation period. The fluctuation in α(2-3)sialyllactose levels afterthe first hour was attributed to the difficulty in obtaining homogenoussamples from the milk cans. Interestingly, a large increase in α(2-3)sialyllactose concentration was observed in the whey after the curddropped from the milk. The end result was α(2-3) trans-sialidasetreatment of milk prior to cheddar cheese production resulted in a 2 to4 fold increase in α(2-3) sialyllactose when compared to other wheysamples the dairy source of which had not been previously treated withα(2-3) trans-sialidase. The addition of α(2-3) trans-sialidase to theraw milk did not have any untoward effect upon the taste or quality ofthe cheese generated using the α(2-3) trans-sialidase treated milk.

Effect of α(2-3) trans-sialidase treatment of milk prior to cheddarcheese production % α(2-3) sialyllactose % Dry α(2-3) sialyllactoseSample (μg/ml) Weight per gram of solid Milk Time = 0  37 12.94 0.02Milk Time = 1 hr 172 10.03 0.17 Milk Time = 2 hr 160 11.52 0.14 MilkTime = 11 hr 181 11.55 0.16 Whey 255  8.91 0.29 Whey 244  7.84 0.31 Whey239  8.42 0.28 Standard dry whey — — 0.06-0.13

5.8 EXAMPLE: ENRICHMENT OF α(2-3) SIALYLLACTOSE IN DAIRY SOURCES ANDCHEESE PROCESSING WASTE STREAMS

This Example investigates the enrichment of α(2-3) sialyllactose indairy sources and cheese processing waste streams that have beencontacted with bacterial lysates containing α(2-3) trans-sialidaseactivity. Bacterial lysates were prepared as set forth infra in Section5.6. Methods for producing and assaying for α(2-3) sialyllactose wereessentially as set forth infra in Section 5.3.

As shown in FIG. 5, the addition of α(2-3) trans-sialidase increasedα(2-3) sialyllactose concentrations over a incubation mixture pH rangeof 4.0-9.0.

Increased α(2-3) sialyllactose concentrations of 2-5 fold were observedin dairy sources and cheese processing waste streams tested including:mozzarella whey (see FIGS. 5 and 7A-7B); skim milk (see FIG. 6); swisscheese whey (see FIG. 8); and in a composition simulating milk (see FIG.9).

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

10 3183 base pairs nucleic acid single linear cDNA not provided 1ATGGGGAAAA CAGTCGTTGG GGCCAGTAGG ATGTTCTGGC TAATGTTTTT CGTGCCGCTT 60CTTCTTGCGC TCTGCCCCAG CGAGCCCGCG CATGCCCTGG CACCCGGATC GAGCCGAGTT 120GAGCTGTTTA AGCGGCAAAG CTCGAAGGTG CCATTTGAAA AGGGCGGCAA AGTCACCGAG 180CGGGTTGTCC ACTCGTTCCG CCTCCCCGCC CTTGTTAATG TGGACGGGGT GATGGTTGCC 240ATCGCGGACG CTCGCTACGA AACATCCAAT GACAACTCCC TCATTGATAC GGTGGCGAAG 300TACAGCGTGG ACGATGGGGA GACGTGGGAG ACCCAAATTG CCATCAAGAA CAGTCGTGCA 360TCGTCTGTTT CTCGTGTGGT GGATCCCACA GTGATTGTGA AGGGCAACAA GCTTTACGTC 420CTGGTTGGAA GCTACAACAG TTCGAGGAGC TACTGGACGT CGCATGGTGA TGCGAGAGAC 480TGGGATATTC TGCTTGCCGT TGGTGAGGTC ACGAAGTCCA CTGCGGGCGG CAAGATAACT 540GCGAGTATCA AATGGGGGAG CCCCGTGTCA CTGAAGGAAT TTTTCCCGGC GGAAATGGAA 600GGAATGCACA CAAATCAATT TCTTGGCGGT GCAGGTGTTG CCATTGTGGC GTCCAACGGG 660AATCTTGTGT ACCCTGTGCA GGTTACGAAC AAAAAGAAGC AAGTTTTTTC CAAGATCTTC 720TACTCGGAAG ACGAGGGCAA GACGTGGAAG TTTGGGGAGG GTAGGAGTGA TTTTGGCTGC 780TCTGAACCTG TGGCCCTTGA GTGGGAGGGG AAGCTCATCA TAAACACTCG AGTTGACTAT 840CGCCGCCGTC TGGTGTACGA GTCCAGTGAC ATGGGGAATT CGTGGGTGGA GGCTGTCGGC 900ACGCTCTCAC GTGTGTGGGG CCCCTCACCA AAATCGAACC AGCCCGGCAG TCAGAGCAGC 960TTCACTGCCG TGACCATCGA GGGAATGCGT GTTATGCTCT TCACACACCC GCTGAATTTT 1020AAGGGAAGGT GGCTGCGCGA CCGACTGAAC CTCTGGCTGA CGGATAACCA GCGCATTTAT 1080AACGTTGGGC AAGTATCCAT TGGTGATGAA AATTCCGCCT ACAGCTCCGT CCTGTACAAG 1140GATGATAAGC TGTACTGTTT GCATGAGATC AACAGTAACG AGGTGTACAG CCTTGTTTTT 1200GCGCGCCTGG TTGGCGAGCT ACGGATCATT AAATCAGTGC TGCAGTCCTG GAAGAATTGG 1260GACAGCCACC TGTCCAGCAT TTGCACCCCT GCTGATCCAG CCGCTTCGTC GTCAGAGCGT 1320GGTTGTGGTC CCGCTGTCAC CACGGTTGGT CTTGTTGGCT TTTTGTCGCA CAGTGCCACC 1380AAAACCGAAT GGGAGGATGC GTACCGCTGC GTCAACGCAA GCACGGCAAA TGCGGAGAGG 1440GTTCCGAACG GTTTGAAGTT TGCGGGGGTT GGCGGAGGGG CGCTTTGGCC GGTGAGCCAG 1500CAGGGGCAGA ATCAACGGTA TCACTTTGCA AACCACGCGT TCACGCTGGT GGCGTCGGTG 1560ACGATTCACG AGGTTCCGAG CGTCGCGAGT CCTTTGCTGG GTGCGAGCCT GGACTCTTCT 1620GGTGGCAAAA AACTCCTGGG GCTCTCGTAC GACGAGAAGC ACCAGTGGCA GCCAATATAC 1680GGATCAACGC CGGTGACGCC GACCGGATCG TGGGAGATGG GTAAGAGGTA CCACGTGGTT 1740CTTACGATGG CGAATAAAAT TGGTTCGGTG TACATTGATG GAGAACCTCT GGAGGGTTCA 1800GGGCAGACCG TTGTGCCAGA CGGGAGGACG CCTGACATCT CCCACTTCTA CGTTGGCGGG 1860TATGGAAGGA GTGATATGCC AACCATAAGC CACGTGACGG TGAATAATGT TCTTCTTTAC 1920AACCGTCAGC TGAATGCCGA GGAGATCAGG ACCTTGTTCT TGAGCCAGGA CCTGATTGGC 1980ACGGAAGCAC ACATGGGCAG CAGCAGCGGC AGCAGTGCCC ACAGTACGCC CTCAACTCCC 2040GCTGACAACG GTGCCCACAG TACGCCCTCA ACTCCCGCTG ACAGCAGTGC CCACAGTACG 2100CCCTCAACTC CCGCTGACAG CAGTGCCCAC AGTACGCCCT CAGCTCCCGG TGACAACGGT 2160GCCCACAGTA CGCCCTCGAC TCCCGGTGAC AGCAGTGCCC ACAGTACGCC CTCAACTCCC 2220GCTGACAACG GTGCCCACAG TACGCCCTCA GCTCCCGCTG ACAGCAATGC CCACAGTACG 2280CCCTCGACTC CCGCTGACAA CGGTGCCCAC AGTACGCCCT CAACTCCCGC TGACAACGGT 2340GCCCACAGTA CGCCCTCGAC TCCCGGTGAC AACGGTGCCC ACAGTACGCC CTCGACTCCC 2400GGTGACAGCA GTGCCCACAG TACGCCCTCA ACTCCCGCTG ACAACGGTGC CCACAGTACG 2460CCCTCAGCTC CCGCTGACAG CAATGCCCAC AGTACGCCCT CGACTCCCGG TGACAACGGT 2520GCCCACAGTA CGCCCTCAGC TCCCGCTGAC AGCAATGCCC ACAGTACGCC CTCGACTCCC 2580GCTGACAGCA GTGCCCACAG TACGCCCTCA GCTCCCGGTG ACAACGGTGC CCACAGTACG 2640CCCTCAGCTC CCGCTGACAG CAGTGCCCAC AGTACGCCCT CAGCTCCCGG TGACAACGGT 2700GCCCACAGTA CGCCCTCAGC TCCCGCTGAC AACGGTGCCC ACAGTACGCC CTCAGCTCCC 2760GGTGACAGCA ATGCCCACAG TACGCCCTCG ACTCCCGCTG ACAGCAGTGC CCACAGTACG 2820CCCTCAACTC CCGCTGACAG CAGTGCCCAC AGTACGCCCT CAGCTCCCGG TGACAACGGT 2880GCCCACAGTA CGCCCTCAGC TCCCGCTGAC AGCAGTGCCC ACAGTACGCC CTCAATTCCC 2940GGTGACAGCA GTGCCCACAG TACGCCCTCA GCTCCCGCTG ACAGCAGTGC CCACAGTACG 3000CCCTCAGCTC CCGGTGACAA CGGTGCCCAC AGTACGCCCT CGACTCCCGC TGACAACGGC 3060GCTAATGGTA CGGTTTTGAT TTTGCACGAT GGCGCTGCAT TTTCGGCCTT TTCGGGCGGA 3120GGGCTTCTTT TGTGTGCGGG TGCTTTGCTG CTGCACGTGT TCGTTATGGC AGTTTTTTTC 3180TGA 3183 1060 amino acids amino acid single linear protein not provided2 Met Gly Lys Thr Val Val Gly Ala Ser Arg Met Phe Trp Leu Met Phe 1 5 1015 Phe Val Pro Leu Leu Leu Ala Leu Cys Pro Ser Glu Pro Ala His Ala 20 2530 Leu Ala Pro Gly Ser Ser Arg Val Glu Leu Phe Lys Arg Gln Ser Ser 35 4045 Lys Val Pro Phe Glu Lys Gly Gly Lys Val Thr Glu Arg Val Val His 50 5560 Ser Phe Arg Leu Pro Ala Leu Val Asn Val Asp Gly Val Met Val Ala 65 7075 80 Ile Ala Asp Ala Arg Tyr Glu Thr Ser Asn Asp Asn Ser Leu Ile Asp 8590 95 Thr Val Ala Lys Tyr Ser Val Asp Asp Gly Glu Thr Trp Glu Thr Gln100 105 110 Ile Ala Ile Lys Asn Ser Arg Ala Ser Ser Val Ser Arg Val ValAsp 115 120 125 Pro Thr Val Ile Val Lys Gly Asn Lys Leu Tyr Val Leu ValGly Ser 130 135 140 Tyr Asn Ser Ser Arg Ser Tyr Trp Thr Ser His Gly AspAla Arg Asp 145 150 155 160 Trp Asp Ile Leu Leu Ala Val Gly Glu Val ThrLys Ser Thr Ala Gly 165 170 175 Gly Lys Ile Thr Ala Ser Ile Lys Trp GlySer Pro Val Ser Leu Lys 180 185 190 Glu Phe Phe Pro Ala Glu Met Glu GlyMet His Thr Asn Gln Phe Leu 195 200 205 Gly Gly Ala Gly Val Ala Ile ValAla Ser Asn Gly Asn Leu Val Tyr 210 215 220 Pro Val Gln Val Thr Asn LysLys Lys Gln Val Phe Ser Lys Ile Phe 225 230 235 240 Tyr Ser Glu Asp GluGly Lys Thr Trp Lys Phe Gly Glu Gly Arg Ser 245 250 255 Asp Phe Gly CysSer Glu Pro Val Ala Leu Glu Trp Glu Gly Lys Leu 260 265 270 Ile Ile AsnThr Arg Val Asp Tyr Arg Arg Arg Leu Val Tyr Glu Ser 275 280 285 Ser AspMet Gly Asn Ser Trp Val Glu Ala Val Gly Thr Leu Ser Arg 290 295 300 ValTrp Gly Pro Ser Pro Lys Ser Asn Gln Pro Gly Ser Gln Ser Ser 305 310 315320 Phe Thr Ala Val Thr Ile Glu Gly Met Arg Val Met Leu Phe Thr His 325330 335 Pro Leu Asn Phe Lys Gly Arg Trp Leu Arg Asp Arg Leu Asn Leu Trp340 345 350 Leu Thr Asp Asn Gln Arg Ile Tyr Asn Val Gly Gln Val Ser IleGly 355 360 365 Asp Glu Asn Ser Ala Tyr Ser Ser Val Leu Tyr Lys Asp AspLys Leu 370 375 380 Tyr Cys Leu His Glu Ile Asn Ser Asn Glu Val Tyr SerLeu Val Phe 385 390 395 400 Ala Arg Leu Val Gly Glu Leu Arg Ile Ile LysSer Val Leu Gln Ser 405 410 415 Trp Lys Asn Trp Asp Ser His Leu Ser SerIle Cys Thr Pro Ala Asp 420 425 430 Pro Ala Ala Ser Ser Ser Glu Arg GlyCys Gly Pro Ala Val Thr Thr 435 440 445 Val Gly Leu Val Gly Phe Leu SerHis Ser Ala Thr Lys Thr Glu Trp 450 455 460 Glu Asp Ala Tyr Arg Cys ValAsn Ala Ser Thr Ala Asn Ala Glu Arg 465 470 475 480 Val Pro Asn Gly LeuLys Phe Ala Gly Val Gly Gly Gly Ala Leu Trp 485 490 495 Pro Val Ser GlnGln Gly Gln Asn Gln Arg Tyr His Phe Ala Asn His 500 505 510 Ala Phe ThrLeu Val Ala Ser Val Thr Ile His Glu Val Pro Ser Val 515 520 525 Ala SerPro Leu Leu Gly Ala Ser Leu Asp Ser Ser Gly Gly Lys Lys 530 535 540 LeuLeu Gly Leu Ser Tyr Asp Glu Lys His Gln Trp Gln Pro Ile Tyr 545 550 555560 Gly Ser Thr Pro Val Thr Pro Thr Gly Ser Trp Glu Met Gly Lys Arg 565570 575 Tyr His Val Val Leu Thr Met Ala Asn Lys Ile Gly Ser Val Tyr Ile580 585 590 Asp Gly Glu Pro Leu Glu Gly Ser Gly Gln Thr Val Val Pro AspGly 595 600 605 Arg Thr Pro Asp Ile Ser His Phe Tyr Val Gly Gly Tyr GlyArg Ser 610 615 620 Asp Met Pro Thr Ile Ser His Val Thr Val Asn Asn ValLeu Leu Tyr 625 630 635 640 Asn Arg Gln Leu Asn Ala Glu Glu Ile Arg ThrLeu Phe Leu Ser Gln 645 650 655 Asp Leu Ile Gly Thr Glu Ala His Met GlySer Ser Ser Gly Ser Ser 660 665 670 Ala His Ser Thr Pro Ser Thr Pro AlaAsp Asn Gly Ala His Ser Thr 675 680 685 Pro Ser Thr Pro Ala Asp Ser SerAla His Ser Thr Pro Ser Thr Pro 690 695 700 Ala Asp Ser Ser Ala His SerThr Pro Ser Ala Pro Gly Asp Asn Gly 705 710 715 720 Ala His Ser Thr ProSer Thr Pro Gly Asp Ser Ser Ala His Ser Thr 725 730 735 Pro Ser Thr ProAla Asp Asn Gly Ala His Ser Thr Pro Ser Ala Pro 740 745 750 Ala Asp SerAsn Ala His Ser Thr Pro Ser Thr Pro Ala Asp Asn Gly 755 760 765 Ala HisSer Thr Pro Ser Thr Pro Ala Asp Asn Gly Ala His Ser Thr 770 775 780 ProSer Thr Pro Gly Asp Asn Gly Ala His Ser Thr Pro Ser Thr Pro 785 790 795800 Gly Asp Ser Ser Ala His Ser Thr Pro Ser Thr Pro Ala Asp Asn Gly 805810 815 Ala His Ser Thr Pro Ser Ala Pro Ala Asp Ser Asn Ala His Ser Thr820 825 830 Pro Ser Thr Pro Gly Asp Asn Gly Ala His Ser Thr Pro Ser AlaPro 835 840 845 Ala Asp Ser Asn Ala His Ser Thr Pro Ser Thr Pro Ala AspSer Ser 850 855 860 Ala His Ser Thr Pro Ser Ala Pro Gly Asp Asn Gly AlaHis Ser Thr 865 870 875 880 Pro Ser Ala Pro Ala Asp Ser Ser Ala His SerThr Pro Ser Ala Pro 885 890 895 Gly Asp Asn Gly Ala His Ser Thr Pro SerAla Pro Ala Asp Asn Gly 900 905 910 Ala His Ser Thr Pro Ser Ala Pro GlyAsp Ser Asn Ala His Ser Thr 915 920 925 Pro Ser Thr Pro Ala Asp Ser SerAla His Ser Thr Pro Ser Thr Pro 930 935 940 Ala Asp Ser Ser Ala His SerThr Pro Ser Ala Pro Gly Asp Asn Gly 945 950 955 960 Ala His Ser Thr ProSer Ala Pro Ala Asp Ser Ser Ala His Ser Thr 965 970 975 Pro Ser Ile ProGly Asp Ser Ser Ala His Ser Thr Pro Ser Ala Pro 980 985 990 Ala Asp SerSer Ala His Ser Thr Pro Ser Ala Pro Gly Asp Asn Gly 995 1000 1005 AlaHis Ser Thr Pro Ser Thr Pro Ala Asp Asn Gly Ala Asn Gly Thr 1010 10151020 Val Leu Ile Leu His Asp Gly Ala Ala Phe Ser Ala Phe Ser Gly Gly1025 1030 1035 1040 Gly Leu Leu Leu Cys Ala Gly Ala Leu Leu Leu His ValPhe Val Met 1045 1050 1055 Ala Val Phe Phe 1060 1929 base pairs nucleicacid unknown linear cDNA not provided 3 ATGCTGGCAC CCGGATCGAG CCGAGTTGAGCTGTTTAAGC GGCAAAGCTC GAAGGTGCCA 60 TTTGAAAAGG ACGGCAAAGT CACCGAGCGGGTTGTCCACT CGTTCCGCCT CCCCGCCCTT 120 GTTAATGTGG ACGGGGTGAT GGTTGCCATCGCGGACGCTC GCTACGAAAC ATCCAATGAC 180 AACTCCCTCA TTGATACGGT GGCGAAGTACAGCGTGGACG ATGGGGAGAC GTGGGAGACC 240 CAAATTGCCA TCAAGAACAG TCGTGCATCGTCTGTTTCTC GTGTGGTGGA TCCCACAGTG 300 ATTGTGAAGG GCAACAAGCT TTACGTCCTGGTTGGAAGCT ACAACAGTTC GAGGAGCTAC 360 TGGACGTCGC ATGGTGATGC GAGAGACTGGGATATTCTGC TTGCCGTTGG TGAGGTCACG 420 AAGTCCACTG CGGGCGGCAA GATAACTGCGAGTATCAAAT GGGGGAGCCC CGTGTCACTG 480 AAGGAATTTT TTCCGGCGGA AATGGAAGGAATGCACACAA ATCAATTTCT TGGCGGTGCA 540 GGTGTTGCCA TTGTGGCGTC CAACGGGAATCTTGTGTACC CTGTGCAGGT TACGAACAAA 600 AAGAAGCAAG TTTTTTCCAA GATCTTCTACTCGGAAGACG AGGGCAAGAC GTGGAAGTTT 660 GGGAAGGGTA GGAGCGCTTT TGGCTGCTCTGAACCTGTGG CCCTTGAGTG GGAGGGGAAG 720 CTCATCATAA ACACTCGAGT TGACTATCGCCGCCGTCTGG TGTACGAGTC CAGTGACATG 780 GGGAATTCGT GGCTGGAGGC TGTCGGCACGCTCTCACGTG TGTGGGGCCC CTCACCAAAA 840 TCGAACCAGC CCGGCAGTCA GAGCAGCTTCACTGCCGTGA CCATCGAGGG AATGCGTGTT 900 ATGCTCTTCA CACACCCGCT GAATTTTAAGGGAAGGTGGC TGCGCGACCG ACTGAACCTC 960 TGGCTGACGG ATAACCAGCG CATTTATAACGTTGGGCAAG TATCCATTGG TGATGAAAAT 1020 TCCGCCTACA GCTCCGTCCT GTACAAGGATGATAAGCTGT ACTGTTTGCA TGAGATCAAC 1080 AGTAACGAGG TGTACAGCCT TGTTTTTGCGCGCCTGGTTG GCGAGCTACG GATCATTAAA 1140 TCAGTGCTGC AGTCCTGGAA GAATTGGGACAGCCACCTGT CCAGCATTTG CACCCCTGCT 1200 GATCCAGCCG CTTCGTCGTC AGAGCGTGGTTGTGGTCCCG CTGTCACCAC GGTTGGTCTT 1260 GTTGGCTTTT TGTCGCACAG TGCCACCAAAACCGAATGGG AGGATGCGTA CCGCTGCGTG 1320 AACGCAAGCA CGGCAAATGC GGAGAGGGTTCCGAACGGTT TGAAGTTTGC GGGGGTTGGC 1380 GGAGGGGCGC TTTGGCCGGT GAGCCAGCAGGGGCAGAATC AACGGTATCG CTTTGCAAAC 1440 CACGCGTTCA CCGTGGTGGC GTCGGTGACGATTCACGAGG TTCCGAGCGT CGCGAGTCCT 1500 TTGCTGGGTG CGAGCCTGGA CTCTTCTGGTGGCAAAAAAC TCCTGGGGCT CTCGTACGAC 1560 GAGAGGCACC AGTGGCAGCC AATATACGGATCAACGCCGG TGACGCCGAC CGGATCGTGG 1620 GAGATGGGTA AGAGGTACCA CGTGGTTCTTACGATGGCGA ATAAAATTGG CTCCGAGTAC 1680 ATTGATGGAG AACCTCTGGA GGGTTCAGGGCAGACCGTTG TGCCAGACGA GAGGACGCCT 1740 GACATCTCCC ACTTCTACGT TGGCGGGTATAAAAGGAGTG ATATGCCAAC CATAAGCCAC 1800 GTGACGGTGA ATAATGTTCT TCTTTACAACCGTCAGCTGA ATGCCGAGGA GATCAGGACC 1860 TTGTTCTTGA GCCAGGACCT GATTGGCACGGAAGCACACA TGGACAGCAG CAGCGACACG 1920 AGTGCCTGA 1929 642 amino acidsamino acid single linear protein not provided 4 Met Leu Ala Pro Gly SerSer Arg Val Glu Leu Phe Lys Arg Gln Ser 1 5 10 15 Ser Lys Val Pro PheGlu Lys Asp Gly Lys Val Thr Glu Arg Val Val 20 25 30 His Ser Phe Arg LeuPro Ala Leu Val Asn Val Asp Gly Val Met Val 35 40 45 Ala Ile Ala Asp AlaArg Tyr Glu Thr Ser Asn Asp Asn Ser Leu Ile 50 55 60 Asp Thr Val Ala LysTyr Ser Val Asp Asp Gly Glu Thr Trp Glu Thr 65 70 75 80 Gln Ile Ala IleLys Asn Ser Arg Ala Ser Ser Val Ser Arg Val Val 85 90 95 Asp Pro Thr ValIle Val Lys Gly Asn Lys Leu Tyr Val Leu Val Gly 100 105 110 Ser Tyr AsnSer Ser Arg Ser Tyr Trp Thr Ser His Gly Asp Ala Arg 115 120 125 Asp TrpAsp Ile Leu Leu Ala Val Gly Glu Val Thr Lys Ser Thr Ala 130 135 140 GlyGly Lys Ile Thr Ala Ser Ile Lys Trp Gly Ser Pro Val Ser Leu 145 150 155160 Lys Glu Phe Phe Pro Ala Glu Met Glu Gly Met His Thr Asn Gln Phe 165170 175 Leu Gly Gly Ala Gly Val Ala Ile Val Ala Ser Asn Gly Asn Leu Val180 185 190 Tyr Pro Val Gln Val Thr Asn Lys Lys Lys Gln Val Phe Ser LysIle 195 200 205 Phe Tyr Ser Glu Asp Glu Gly Lys Thr Trp Lys Phe Gly LysGly Arg 210 215 220 Ser Ala Phe Gly Cys Ser Glu Pro Val Ala Leu Glu TrpGlu Gly Lys 225 230 235 240 Leu Ile Ile Asn Thr Arg Val Asp Tyr Arg ArgArg Leu Val Tyr Glu 245 250 255 Ser Ser Asp Met Gly Asn Ser Trp Leu GluAla Val Gly Thr Leu Ser 260 265 270 Arg Val Trp Gly Pro Ser Pro Lys SerAsn Gln Pro Gly Ser Gln Ser 275 280 285 Ser Phe Thr Ala Val Thr Ile GluGly Met Arg Val Met Leu Phe Thr 290 295 300 His Pro Leu Asn Phe Lys GlyArg Trp Leu Arg Asp Arg Leu Asn Leu 305 310 315 320 Trp Leu Thr Asp AsnGln Arg Ile Tyr Asn Val Gly Gln Val Ser Ile 325 330 335 Gly Asp Glu AsnSer Ala Tyr Ser Ser Val Leu Tyr Lys Asp Asp Lys 340 345 350 Leu Tyr CysLeu His Glu Ile Asn Ser Asn Glu Val Tyr Ser Leu Val 355 360 365 Phe AlaArg Leu Val Gly Glu Leu Arg Ile Ile Lys Ser Val Leu Gln 370 375 380 SerTrp Lys Asn Trp Asp Ser His Leu Ser Ser Ile Cys Thr Pro Ala 385 390 395400 Asp Pro Ala Ala Ser Ser Ser Glu Arg Gly Cys Gly Pro Ala Val Thr 405410 415 Thr Val Gly Leu Val Gly Phe Leu Ser His Ser Ala Thr Lys Thr Glu420 425 430 Trp Glu Asp Ala Tyr Arg Cys Val Asn Ala Ser Thr Ala Asn AlaGlu 435 440 445 Arg Val Pro Asn Gly Leu Lys Phe Ala Gly Val Gly Gly GlyAla Leu 450 455 460 Trp Pro Val Ser Gln Gln Gly Gln Asn Gln Arg Tyr ArgPhe Ala Asn 465 470 475 480 His Ala Phe Thr Val Val Ala Ser Val Thr IleHis Glu Val Pro Ser 485 490 495 Val Ala Ser Pro Leu Leu Gly Ala Ser LeuAsp Ser Ser Gly Gly Lys 500 505 510 Lys Leu Leu Gly Leu Ser Tyr Asp GluArg His Gln Trp Gln Pro Ile 515 520 525 Tyr Gly Ser Thr Pro Val Thr ProThr Gly Ser Trp Glu Met Gly Lys 530 535 540 Arg Tyr His Val Val Leu ThrMet Ala Asn Lys Ile Gly Ser Glu Tyr 545 550 555 560 Ile Asp Gly Glu ProLeu Glu Gly Ser Gly Gln Thr Val Val Pro Asp 565 570 575 Glu Arg Thr ProAsp Ile Ser His Phe Tyr Val Gly Gly Tyr Lys Arg 580 585 590 Ser Asp MetPro Thr Ile Ser His Val Thr Val Asn Asn Val Leu Leu 595 600 605 Tyr AsnArg Gln Leu Asn Ala Glu Glu Ile Arg Thr Leu Phe Leu Ser 610 615 620 GlnAsp Leu Ile Gly Thr Glu Ala His Met Asp Ser Ser Ser Asp Thr 625 630 635640 Ser Ala 30 base pairs nucleic acid unknown unknown Other notprovided 5 TTTTCTAGAA TGCTGGCACC CGGATCGAGC 30 30 base pairs nucleicacid unknown unknown Other not provided 6 CTGTGCGACA AAAAGCCAACAAGACCAACC 30 29 base pairs nucleic acid unknown unknown Other notprovided 7 ACTGAACCTC TGGCTGACGG ATAACCAGC 29 30 base pairs nucleic acidunknown unknown Other not provided 8 TTTCTCGAGT CAGGCACTCG TGTCGCTGCT 3040 base pairs nucleic acid unknown unknown Other not provided 9GGGCAAGTAT CCATTGGTGA TGAAAATTCC GCCTACAGCT 40 40 base pairs nucleicacid unknown unknown Other not provided 10 TACAGCTTAT CATCCTTGTACAGGACGGAG CTGTAGGCGG 40

What is claimed is:
 1. A method for enriching α(2,3) sialyllactose in adairy source selected from the group consisting of milk, colostrum and acheese processing mixture, comprising: (i) contacting a catalytic amountof at least one α(2,3)trans-sialidase with the dairy source, wherein thedairy source comprises lactose and sialic acid donors to form adairy/trans-sialidase mixture; and (ii) incubating saiddairy/trans-sialidase mixture under conditions suitable to effect theα(2,3)trans-sialidase-catalyzed transfer of sialic acid from the sialicacid donors to the lactose.
 2. The method of claim 1 wherein the α(2-3)trans-sialidase is a Kinetoplastid trans-sialidase.
 3. The method ofclaim 1 wherein the α(2-3) trans-sialidase is encoded by a gene isolatedfrom a species of the genera selected from the group consisting ofTrypanosoma, Endotrypanum and Pneumocystis.
 4. The method of claim 1wherein the α(2-3) trans-sialidase is recomnbinantly produced.
 5. Themethod of claim 1 wherein the dairy source/trans-sialidase mixture isincubated for at least 1 hour.
 6. The method of claim 1 wherein thedairy source/trans-sialidase mixture is incubated at a temperature ofabout 5° C. to about 45° C.
 7. The method of claim 1 wherein the dairysource/trans-sialidase mixture has a pH of about 6 to about
 8. 8. Themethod of claim 1 further comprising recovering α(2-3) sialyllactosefrom the dairy/trans-sialidase mixture.
 9. The method of claim 8 whereinthe recovering step comprises ultrafiltration of the incubateddairy/trans-sialidase mixture to form an ultrafiltrate.
 10. The methodof claim 9 wherein the recovery step further comprises contacting saidultrafiltrate with an ion exchange resin.
 11. The method of claim 10wherein the ion exchange resin is an anion exchange resin.
 12. Themethod of claim 10 wherein the ion exchange resin is a cation exchangeresin.
 13. The method of claim 8 wherein the recovering step comprises:(a) contacting said incubated dairy source/trans-sialidase mixture ofstep (ii) with a solvent and extracting the α(2-3) sialyllactose withsaid solvent to form a α(2-3) sialyilactose containing solvent; (b)separating said α(2-3) sialyllactose containing solvent from saidincubated dairy source/trans-sialidase mixture; and (c) isolating saidα(2-3) sialyllactose from said α(2-3) sialyllactose containing solvent.14. A method for enriching α(2-3) sialyllactose in a cheese processingwaste stream comprising: (i) contacting a catalytic amount of at leastone α(2-3) trans-sialidase with a cheese processing waste stream,wherein the cheese processing waste stream comprises lactose and sialicacid donors to form a waste streamltrans-sialidase mixture; and (ii)incubating said waste stream/trans-sialidase mixture under conditionssuitable to effect the α(2-3) trans-sialidase-catalyzed transfer ofsialic acid from the sialic acid donors to the lactose.
 15. The methodof claim 14 wherein said α(2-3) trans-sialidase is a Kinetoplastidtrans-sialidase.
 16. The method of claim 14 wherein said α(2-3)trans-sialidase is encoded by a gene isolated from a species of thegenus Trypanosoma, Endotiyanum, or Pneumocystis.
 17. The method of claim14 wherein said α(2-3) trans-sialidase is recombinantly produced. 18.The method of claim 14 wherein the waste stream/trans-sialidase mixtureis incubated for at least 1 hour.
 19. The method of claim 14 wherein thewaste stream/trans-sialidase mixture is incubated at a temperature ofabout 5° C. to about 45° C.
 20. The method of claim 14 wherein the wastestream/trans-sialidase mixture has a pH of about 5 to about
 8. 21. Themethod of claim 14 wherein the cheese processing waste stream comprisesa member selected from the group consisting of: whole whey,demineralized whey permeate, the regeneration stream from demineralizedwhey permeate, whey permeate, whey powder and lactose.
 22. The method ofclaim 14 further comprising recovering α(2-3) sialyllactose from saidincubated waste stream/trans-sialidase mixture.
 23. The method of claim22 wherein the recovering step comprises ultrafiltration of theincubated waste stream/trans-sialidase mixture to form an ultrafiltrate.24. The method of claim 23 wherein the recovering step further comprisescontacting said ultrafiltrate with an ion exchange resin.
 25. The methodof claim 24 wherein the ion exchange resin is an anion exchange resin.26. The method of claim 25 wherein the ion exchange resin is a cationexchange resin.
 27. The method of claim 22 wherein the recovering stepcomprises: (a) contacting said incubated waste stream/trans-sialidasemixture of step (ii) with a solvent and extracting said α(2-3)sialyllactose containing solvent; (b) separating said α(2-3)sialyllactose containing solvent from said incubated wastestream/trans-sialidase mixture; and (c) isolating said α(2-3)sialyllactose from said α(2-3) sialyllactose containing solvent.
 28. Themethod of claim 13 or 27 wherein said solvent is selected from the groupconsisting of water C₁-C₅ alcohol and a mixture thereof.
 29. The methodof claim 14 wherein said cheese processing waste stream is the motherliquor obtained by crystallizing lactose from whey.
 30. The method ofclaim 1 or 14 wherein exogenous α(2-3) sialyloligosaccharides are addedduring said incubating step. 31.The method of claim 1 further comprisingthe step of processing the dairy/trans-sialidase mixture forcheesemaking before the recovery step.
 32. The method of claim 21wherein the lactose is crystallized lactose, spray dried lactose, oredible lactose.
 33. The method of claim 1 or 14 wherein the sialic aciddonors are α(2-3) sialosides.
 34. The method of claim 33 wherein theα(2-3) sialosides are selected from the group consisting of κ casein,gangliosides and mixtures thereof.
 35. The method of claim 1 or 14wherein the sialic acid donors are sialyl-α(2-3)-β-galactosides.